N-acetyllactosamine-enriched glycoconjugates and sequence similarities in the carbohydrate recognition domain. Galectin-1, a member of this family, contributes to different events associated with cancer biology, including tumour transformation, cell cycle regulation, apoptosis, cell adhesion, migration and inflammation. In addition, recent evidence indicates that galectin-1 contributes to tumour evasion of immune responses. Given the increased interest of tumour biologists and clinical oncologists in this field and the potential use of galectins as novel targets for anticancer drugs, we summarise here recent advances about the role of galectin-1 in different events of tumour growth and metastasis.Galectins are a family of structurally related carbohydrate-binding proteins, which are defined by their affinity for poly- As they can bind to extracellular glycoconjugates, galectins might modulate the adhesion between adjacent cancer cells or between cancer cells and ECM. It has been shown that galectin-1 increases the adhesion of prostate and ovarian cancer cell lines to the ECM  and it is uncertain whether high levels of soluble protein can be achieved  the ECM .N-acetyllactosamine ligands, may determine T-cell susceptibility to galectin-1-induced cell death  and interferon-γ (IFN-γ) by activated T cells, with no evidence of T-cell apoptosis  provides a stop signal for T-cell adhesion to ECM and abrogates the production of proinflammatory cytokines, such as tumour necrosis factor-β (TGF-β), interleukin 10 (IL-10) and vascular endothelial growth factor (VEGF).Despite the existence of specific T lymphocytes recognising tumour cells, the impact of these cells in tumour growth has been so far elusive. In contrast, several mechanisms have been described that potentially contribute to tumour cell evasion of the immune response  and stimulated the generation of a tumour-specific T-cell response in vivo. Our observations support the idea that galectin-1 may contribute to immune privilege of tumours by modulating survival or polarisation of effector T cells, and suggest a potential molecular target for manipulation of T-cell apoptosis with potential implications in immunotherapy.The immunoregulatory effects of galectin-1 and the correlation between galectin-1 expression in cancer cells and the aggressiveness of these tumours prompted us to investigate the role of galectin-1 in tumor-immune escape. We hypothesised that tumour cells may impair T-cell effector functions through secretion of galectin-1 and that this mechanism may contribute in tilting the balance towards an immunosuppressive environment at the tumour site. By a combination of Given the contribution of galectin-1 to tumour growth and metastasis, it is predicted that inhibitors of galectin-1 will find their way into cancer clinical trials, leading to delays in tumour progression and improvements in overall survival. Challenges for the future will be to employ potent and selective small inhibitors of galectin-1 and, in fact, molecules with such properties have already been developed for galectin-1 or other galectins (


GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from −/−Gimap5 BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in −/−Gimap5 T cells. Knockdown of CHOP by siRNA protected −/−Gimap5 T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human  GIMAP5, has an important role in human immune modulation as a polyadenylation polymorphism in the human GIMAP5 gene increases systemic lupus erythematosus risk gimap5 null mice Gimap5 gene in vitro, and severe T cell lymphopenia in vivoin vitro recapitulates the natural apoptosis that occurs in T cells from the BBDP rat Gimap5 promotes T cell survival is not understood.The expression products of the GTPase of the immunity-associated gene family  have been implicated in the regulation of T cell survival through modulation of T cell receptor (TCR) signaling 2+) signaling regulation, vesicle trafficking, drug metabolism, and lipid biogenesis 2+ homeostasis Gimap5 has been reported to localize mainly to the endoplasmic reticulum (ER) Normally, stress within the ER triggers an adaptive cellular mechanism known as the unfolded protein response or ER stress response that attempts to return the cell to homeostasis chop (C/EBP-homologous protein) bcl-2 gene, thus downregulating antiapoptotic Bcl-2 protein and rendering cells sensitive to the proapoptotic effects of BH3-only proteins −/−Gimap5 T cells as evidenced by an increase in ER chaperone expression. We further show that initiation of ER stress-induced apoptotic signaling in −/−Gimap5 T cells is associated with an increase of CHOP protein. Knockdown of CHOP expression protects −/−Gimap5 T cells from ER stress-induced cell death, thus revealing an essential role for Gimap5 in the maintenance of ER homeostasis and T cell survival.Initiation of ER stress-induced apoptosis through ER stress response signaling involves transcriptional activation of the bZIP transcription factor −/−Gimap5 rats, we compared the expression of ER chaperone proteins in lymph node cells and thymocyte lysates from −/−Gimap5 BBDP and +/+Gimap5 BioBreeding diabetes-resistant (BBDR) rats. Western blot analyses revealed that GRP94, GRP78, and ER protein 72 (ERp72) levels were increased in lymphocytes from −/−Gimap5 BBDP rats when compared to that in +/+Gimap5 BBDR rats  as compared to Gimap5+/+ BBDR rats  in BBDP (69.1±0.7%) vs BBDR rats  was significantly less than in the +/+Gimap5 BBDR rats , whereas B cell numbers were significantly increased . To determine the T cell-specific levels of ER stress response and ER stress-induced apoptotic signaling, we performed intracellular flow cytometry. As measured by the mean fluorescence intensity (MFI), both GRP78 and CHOP levels were higher in TCR+ lymphocytes from −/−Gimap5 BBDP rats than from +/Gimap5+ BBDR rats (−CD45RA+ cells) exhibited no difference in GRP78 and CHOP expression between the two rat strains  was reduced to a much greater degree than was the CD4+ subset  . Absolute CD8+ T cell number in lymph node cells were 1.4±0.1×106 in the BBDP rat vs 30.6±4.5×106 in the BBDR, .We next examined ER stress response activation within peripheral CD4, n = 4) . Absolut+ subset, we again analyzed GRP78 and CHOP by intracellular flow cytometry. We observed that GRP78 and CHOP expression was similar in both CD4+ and CD8+ T cells from the −/−Gimap5 BBDP rat, but both subsets of BBDP T cells had GRP78 and CHOP levels that were higher than their counterparts from the +/+Gimap5 BBDR rat . Flow cytometry demonstrated that the preponderance of CD4+ and CD8+ T cells from −/−Gimap5 BBDP rats still express CD90 on the cell surface, indicating that few T cells survive to the mature CD90− stage  and CD8+  RTEs and mature CD4+  and CD8+  T cells from the −/−Gimap5 BBDP rat that express CD25, a marker of activation (see + and CD8+ RTEs and mature T cells (CD90−) that express CD25 have significantly increased GRP78 expression as compared to cells with the same phenotype in the BBDR rat , early apoptotic , and late apoptotic and necrotic   . As compuadrant) . In contuadrant) .− lymphocytes. In contrast to T cells, there were no statistical differences in the percentages of viable, early apoptotic, and late apoptotic TCR− lymphocytes between siCHOP and siControl transfected cells .The intracellular distribution of Gimap5 protein remains controversial, but recent research indicates localization to the ER Gimap5 which results in a truncated protein product −/−Gimap5 T cells. In either case, disruption of CHOP was effective in delaying apoptosis and preventing cell death, thus indicating the importance of CHOP in ER stress-associated T cell death signaling. Interestingly, overexpression of either wild type rat Gimap5 or the truncated BBDP rat Gimap5 in a rat T cell line is associated with enhanced apoptosis In the BBDP rat there is the frameshift mutation in −/−Gimap5 rats. It will be of interest to determine if the same mechanisms are responsible for the T cell lymphopenia and hepatic dysfunction observed in −/−gimap5 mice In conclusion, our findings suggest a role for ER stress in mediating the spontaneous apoptosis found in T cells from −/−Gimap5 BBDP and +/+Gimap5 BBDR rats were obtained from Biomedical Research Models . BBDP rats are T cell lymphopenic in both the CD4 and to an even greater extent in the CD8 compartments; BBDR rats have a normal peripheral lymphocyte phenotype −/−ART2a.Gimap5 rats were also developed by us by crossing WF  and BBDP rats. The intercross was followed by 8 backcrosses to WF before intercrossing to establish homozygosity for both the Iddm14lyp (Gimap5) loci on chromosome 4 and the ART2a locus −/−ART2a.Gimap5 rat bears no BB markers except in the regions of chromosome 4 bounded by D4Rat101-D4Rat58 and chromosome 1 bounded by D1Rat29 and D1Rat287, and it does not develop spontaneous diabetes (unpublished observations). Control WF.ART2a congenic rats were developed by us and differ from ordinary non-lymphopenic, non-diabetic WF animals in that they express the “a” rather than the “b” allotype of the ART2 T cell alloantigen on chromosome 1 ART2a are nondiabetic and homozygous for wild type Gimap5. All animals were housed in a viral antibody-free facility and maintained in accordance with the guidelines of the University of Massachusetts Medical School Institutional Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals .Thymi and cervical and mesenteric lymph nodes were removed from BBDR and BBDP rats, processed aseptically, and extruded through mesh sieves. Single cell suspensions for flow cytometry were prepared as described below.Thymi, lymphocytes, or purified T cells were lysed Anti-rat CD8a-PE (clone Ox-8), FITC or PerCp-conjugated anti-rat TCRαβ (clone R73), anti-rat CD90-FITC (clone HIS51), Biotin-conjugated anti-rat CD25 (clone OX-39), and Streptavidin-conjugated APC-Cy7 mAbs were obtained from BD Biosciences . Purified GRP78 mAbs (clone 40) and isotype control Abs were also obtained from BD Biosciences. A Zenon Mouse IgG2a or IgG1 Labeling Kit, Alexa Fluor 647 , was used to label purified GRP78 or CHOP mAbs per the manufacturer's directions. Anti-rat CD4-Pacific Blue mAb and its corresponding isotype control Ab were obtained from Serotec . Anti-actin (clone C4) antibody was obtained from Chemicon International . Anti-ERp72 and anti-GRP94 were obtained from Calbiochem  and Stressgen , respectively. Cleaved capase-3 antibody was obtained from Cell Signaling Technology . Polyclonal antiserum to Gimap5 was generated by us as described Single cell suspensions from cervical and mesenteric lymph node cells were washed and suspended in PBS containing 1% fetal clone serum  and 0.1% sodium azide (Sigma-Aldrich). Samples were washed, incubated for 20 min with fluorescent mAbs to various cell-surface markers as described in 6 cells per well in 3 mL of RPMI medium (Sigma-Aldrich) containing 10% FBS (Hyclone), 1% Pen/Strep/Glut , and 0.1% β-mercaptoethanol (Gibco) at 37°C in the presence of 10 ng/mL PMA  and 100 ng/mL ionomycin . After 12 h, cells were resuspended in 100 µL of Nucleofector solution using human T cell Nucleofector kit  with 100 nM rat CHOP siRNA  or 100 nM control siRNA  and nucleofected using program U-014 in the Amaxa Nucleofector apparatus. Following nucleofection, T cells were plated in 3 mL complete media and harvested at 30 h for protein preparation and flow cytometry analysis.T cells from BBDP rats were purified as described To measure cellular apoptotic signaling, transfected T cells were harvested, washed twice with PBS, and reacted with PE-conjugated anti-annexin V and 7-amino-actinomycin D (7AAD) according to the manufacturer's directions (BD Pharmingen). At least 20,000 cells per sample were analyzed using a FACSCalibur (BD Biosciences) and FlowJo software.t-tests. Values of p<0.05 were considered statistically significant.Statistical analyses were performed with GraphPad Prism software . Parametric data are presented as the mean±the standard error of measurement (SEM) and were analyzed using two-tailed unpaired Figure S1−/−ART2a.Gimap5 rats that do not develop spontaneous diabetes exhibit enhanced ER stress response signaling. Representative analyses indicating the gating of TCR+ (A) and CD4+ or CD8+ (B) lymphocytes within congenic WF.−/−ART2a.Gimap5 (Type 31) and control congenic WF.+/+ART2a.Gimap (Type 20) rats. Representative flow dot plots depicting CD90 expression  and forward scattering  of lymphocytes within gated CD4+ and CD8+ T cell populations from Type 31 (C) or Type 20 (D) rats. Numbers represent the percentage of cells (±SEM of triplicate samples) in each gate shown. Intracellular GRP78 (E) and CHOP (F) expression in RTEs (CD90+) and mature T cells (CD90−) from Type 31 (black line) and Type 20 rats (shaded region). Depicted in each histogram is the isotype control (dotted line). The MFI of GRP78 or CHOP protein expression is displayed in bar graphs with error bars representing the SEM of duplicate samples. Data shown are representative of two independent experiments .T cell populations from congenic WF.(0.94 MB TIF)Click here for additional data file.


The differential level and localization of galectin-3 protein were examined in the retinas of two-day-old pigs and six-month-old pigs.The retinas sampled from two-day-old and six-month-old pigs were analyzed by western blot and immunohistochemistry.western blot analysis detected galectin-3 in both age groups, although the levels were significantly higher in six-month-old pigs. Immunohistochemical staining showed that galectin-3 was localized in the retinas of both two-day-old pigs and six-month-old pigs; the galectin-3 immunostaining was more intense in the six-month-old pig retina, as shown in the western blot analysis. Galectin-3 was expressed in glial cells, particularly in glutamine synthetase-positive Müller cells and their processes, across all retina layers in both age groups; however, it was not found in ganglion cells of the ganglion cell layer or neuronal cells of the inner and outer nuclear cell layers in either age group.This is the first demonstration that galectin-3 is detected in the retinas of two-day-old pigs and that the expression in Müller cells increases with postnatal development. Galectin-3, which has a carbohydrate-recognition domain and an N-terminal domain rich in proline and glycine, is characterized by its unique structure among vertebrate galectins . FifteenThe retina is remote CNS tissue and is a model for studying CNS tissue development. The pig eye has been suggested as a novel model for human eye disease because the pig retina shares many similarities with the human retina -17. The The present study examined the expression and cellular localization of galectin-3 in the retinas of two-day-old and six-month-old pigs to evaluate the differential level and localization of galectin-3 during postnatal development in the pig retinas.2 inhalation. The anterior segment of the eyeball was removed, and the retinas were carefully dissected on ice. All the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals in Jeju National University, Jeju, Korea.The eyes of six-month-old young adult pigs (n=5) were obtained immediately after butchering in a local slaughterhouse. Two-day-old pigs were obtained from a local farm (n=3). These were killed by CO2PO4, 1.4mM KH2PO4, pH 7.4) for 24 h. Chemicals for PBS were obtained from JUNSEI . Fixed tissues were dehydrated through a graded series of ethanol and embedded in tissue embedding medium . The paraffin-embedded retinal tissues were cut 5μm on a rotary microtome .For western blot analysis, retinal tissues were detached from the eyecups and kept in a deep freezer. For histology, the anterior segments of the eyes were dissected. The eyecups without anterior segments were fixed by immersion in 10% neutral-buffered formalin in phosphate-buffered saline . Mouse monoclonal anti-glial fibrillary acidic protein  was used to detect astrocytes while anti-glutamine synthetase  was employed to detect Müller cells [Approximately 1 mg/ml rat anti-galectin-3 monoclonal antibody was purified by affinity chromatography from the supernatants of hybridoma cells  for 2 h at 100 V. The residual binding sites on the membrane were blocked by incubation for 1 h with 5% nonfat milk in Tris-buffered saline (TBS), which was composed of 10 mM Tris-HCl, pH 7.4, and 150 mM NaCl. The membranes were then incubated for 2 h either with 1:5,000 rat monoclonal anti-galectin-3 or 1:20,000 mouse monoclonal anti-glutamine synthetase  for 2 h. The blots were washed three times in TBS containing 0.1% Tween-20 and probed with horseradish peroxidase-conjugated anti-rat IgG  for 1 h. The membrane blots were developed using an enhanced chemiluminescence reagent kit , according to the manufacturer’s instructions.Western blot analysis of the pig retina was performed as reported previously, with minor modifications ,22. In b2) of each band was measured with a scanning laser densitometer  and was reported as the mean±SEM. The ratios of the density of the galectin-3 band to that of the β-actin band were compared using Molecular Analyst software (Bio-Rad). The results were analyzed statistically using one-way ANOVA (ANOVA) followed by the post hoc Student–Newman–Keuls t-test for multiple comparisons. In all cases, a p<0.05 was considered significant.After imaging, the membranes were stripped and reprobed using monoclonal anti-β-actin antibody as the primary antibody (Sigma-Aldrich). The optical density (OD/mmParaffin sections (5 μm) of retinas were deparaffinized in xylene, rehydrated in a series of decreasingly diluted solutions of ethanol and pretreated with 0.01 M citrate buffer (pH 6.0) in a microwave for 3 min. After hydration, the sections were treated with 0.3% hydrogen peroxide in distilled water for 20 min to block endogenous peroxidase activity. After three washes in PBS, the sections were exposed to 10% normal goat serum  and then incubated for 1 h at room temperature (RT) with primary antibodies, including 1:500 monoclonal rat anti-galectin-3. After three washes, the sections were incubated with the secondary antibody, 1:100 biotinylated rabbit anti-rat IgG (Vector), and then avidin-biotin peroxidase complexes were formed using an Elite kit (Vector). The peroxidase reaction was developed with a diaminobenzidine substrate kit (Vector). Before sections were mounted, they were counterstained with hematoxylin. As a negative control, the primary antiserum was omitted for a few test sections in each experiment, and no specific labeling of cell bodies or fibers was detected in these sections.2O, treated them with 10 mM CuSO4 in ammonium acetate buffer  for 20 min, then again dipped them in distilled H2O before returning them to PBS. The double immunofluorescence-stained specimens were examined under an FV500 laser confocal microscope .For double immunofluorescence, the sections were incubated in the following order: with 10% normal goat serum for 1 h at RT, with either mouse monoclonal anti-GFAP (Sigma-Aldrich) or mouse monoclonal anti-glutamine synthetase (Chemicon) for 1 h, and then with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Sigma-Aldrich) for 1 h at RT. Then, the sections were washed and incubated with rat anti-galectin-3 overnight at 4 °C, after which the sections were washed and incubated with tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rat IgG (Sigma-Aldrich). To reduce or eliminate lipofuscin autofluorescence, we washed the sections in PBS. Three times for 1 h at RT, dipped them briefly in distilled HWestern blot analysis was used to detect galectin-3  in the homogenates of retinas from two-day-old pigs and six-month-old pigs . CompareIn the retinas of two-day-old piglets, galectin-3 immunostaining was observed in glial processes in the innermost nerve fiber layer and the ganglion cell layer . It was A double-labeling experiment was performed to determine the cellular phenotype of galectin-3 expression in the pig retina. The results for six-month-old pig retinas are shown because the galectin-3 immunoreactivity was similar in both age groups. Galectin-3 was colocalized with glutamine synthetase, a marker for Müller cells , in the This is the first study to demonstrate that galectin-3 is expressed in the pig retina and that its expression increases with development. The expression of both galectin-3 and glutamine synthetase was more intense in the retinas of six-month-old pigs than in those of two-day-old pigs. This suggests that galectin-3 is detected in the retinas of postnatal pigs, and that the increased expression of galectin-3 in the adult retina is correlated with retinal development with aging. The functional roles of increased galectin-3 during retinal development are not clear. It is possible that galectin-3 plays a role in cellular protection, because the interaction of galectin-3 with a glycoconjugate on the cell surface could increase cell viability and anti-apoptotic activity .A previous study has shown that the pig retina is mature one week before birth, with the exception of the photoreceptor segments, which are still differentiating . This fiUehara et al.  detected


However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine . The prepared PCFC-g-PEI was characterized by 1H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).The cytotoxicity and  Gene therapy is being studied world-widely during the past 20 years. In almost all of experiments and clinical treatments, gene therapy requires delivering therapeutic gene into target cells to correct gene defects and achieve the purpose of treating diseases by using delivery carriers. People have never stopped pursuing more safe and efficient vector for gene delivery in gene therapy ,2. Gene in vitro transfection efficiency and enhance the transgene expression in vivo /[pDNA] N/P ratios and shortly vortexed. After 10 min incubation at room temperature, 10 μl complex solutions were analyzed by 1% agarose gel electrophoresis . In the part of protection and release assay of DNA, PCFC-g-PEI/DNA complexes were analyzed using atomic force microscopy (AFM). The complex solution was prepared by mixing 2 μg of plasmid DNA with polymer solution at N/P ratio 7. The complex solution was incubated at room temperature for 30 min and diluted in 1 ml distilled water, and then the complex solution was placed on mica surface and dried at temperature. Imaging was carried out using noncontact mode, and complexes sizes were determined by measuring the diameters of the complexes.The morphology and size of the PCFC-2 and passage every 2–3 days. The cells (1 × 105 per well) were plated on 6-well plated for 24 h before transfection. Immediately before the initiation of transfection experiments, the medium was removed from each well and washed once with DMEM without serum and antibiotic and treated with fresh medium with a serial dilution of PCFC-g-PEI (25 kD). PEI/DNA complex was performed as control. Luciferase gene was monitored 24 h later by using Flow Cytometry (FCM).For in vitro transfection, 293 T and A549 cells were cultured in DMEM medium containing Gln supplemented with 10% heated-inactivated fetal bovine serum and antibiotics . Cells were grown at 37°C in humidified air containing 5% COg-PEI was determined by MTT analysis and propidium iodide (PI) staining and flow cytometry.The cell cytotoxicity of the PCFC-4 (293 T) and 2 × 104 (A549) cells/well in 0.1 ml growth medium and incubated overnight, and then added 0.1 ml fresh DMEM growth medium containing a series of concentrations of PCFC-g-PEI to each well. Untreated cells in growth media were used as a control. Cell were incubated for 24 h and then followed by addition of 20 μl of 3--2, 5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml). After further incubation for 4 hours, the MTT solution (0.5 mg/ml) was carefully removed from each well, and 150 μl DMSO was added to dissolve the MTT formazan crystals. The absorbance was recorded at 570 nm by an ELISA microplate reader (Bio-Rad). The cell viability (%) was related to the control wells containing untreated cells with fresh cell culture medium and was calculated according to the following:In the part of MTT assay, Cells were seeded in 96-well plates at a density of 1 × 10All dates are presented as the mean of there measurements (± SD).5 (293 T) cells/well in 2 ml growth medium and incubated overnight at 37°C under 5% CO2 until the confluency reached 50~60%, and then changed the medium with 2 ml fresh DMEM growth medium containing various concentrations of PCFC-g-PEI to each well. Untreated cells in growth media were used as a control. Cell were incubated for 24 h and then collected for analysis. Survival (%) = 1 - apoptosis (%).In the part of propidium iodide (PI) staining and flow cytometry, cells were seeded in 6-well plates at a density of 2 × 10PEI: polyethyleneimine; PCFC: poly (ε-caprolactone)-pluronic- poly(ε-caprolactone); F105: poly (ethylene glycol)-poly (propylene glycol)-poly (ethylene glycol); GMA: glycidyl methacrylate 97%; DMEM: Dulbecco's Modified Eagle's Medium; MTT: 3--2, 5-diphenyltetrazolium bromide; DMSO: dimethyl sulfoxide; MW: molecular weight.The authors declare that they have no competing interests.QZY, WYQ and ZX designed the experiments. And the research funds were supported by QZY and WYQ. QZY and LF corrected the manuscript. SS and GQF carried out experiments, analyzed the data, and wrote the manuscript; GQF and FSZ participated in the synthesis of the PCFC-g-PEI copolymer. GCY and DHX analyzed data and revised the manuscript. KB and WXH participated in the MTT cytotoxicity study.All authors read and approved the final manuscript.


Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG. Prostate cancer is the second most prevalent cancer among men in the United States and ranks second to lung cancer in terms of annual mortality . Microarin vitro and in animal models  family of receptors and their ligands in regulating AR activity during prostate cancer progression is currently a focus of intense investigation. This receptor family includes four members: EGFR (ErbB1), ErbB2 , ErbB3 (Her3) and ErbB4 (Her4). All EGFR family members contain an extracellular ligand binding domain, a transmembrane region important in regulating receptor activity, and a cytoplasmic tyrosine kinase domain. ErbB3 lacks tyrosine kinase activity due to amino-acid substitutions in the conserved kinase domain . ErbB rein vitro and in vivo to stimulate growth of prostate cancer cells. For example, in vitro AR is activated in a ligand independent manner by EGF  .All cell lines except PC-3 AR were obtained from the American Type Culture Collection . PC-3 AR cells (ebp1 expression construct has been previous described (The PSA reporter luciferase construct was a gift from Dr Martin Gleave and contains −630/+12 of the 5′ PSA flanking region. The Probasin (−285/+32) luciferase reporter and the pSG5-hAR expression construct were gifts of Dr O Janne. The MMTV-luciferase plasmid was obtained from Dr Joseph Fondell . The ebp−1 of HRG β1  for the indicated times. Cell lysates were prepared and immunoprecipitated as described previously (M HEPES (pH 7.5), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100 and Complete™ protease inhibitor. Protein concentrations were measured using a detergent compatible kit . Cell lysates were precleared with Protein A/Protein G agarose and immunoprecipitated for 4 h at 4°C with 2 μg of a monoclonal antibody directed against ErbB3  and 20 μl packed Protein A/G agarose beads. The immunoprecipitates were washed and resuspended in Laemmli sample buffer. Proteins were resolved by SDS–PAGE. After electrophoresis, the proteins were transferred onto Immobilin-P membranes, and immunoblotted as described  for 24 h. Cells were then stimulated with or without HRG β1  for 1 h. Cell lysates were immunoprecipitated as described above using the polyclonal antibody to Ebp1. Western blot analysis was performed using a monoclonal antibody to AR (Santa Cruz) or the Ebp1 antibody.To measure ErbB3–Ebp1 interactions, LNCaP cells were incubated overnight in serum-free RPMI-1640 media. Where indicated, cells were treated with 20 ng ml4) were plated in 12-well plates in complete media. When cells reached 50–60% confluence, they were transfected using the Fugene-6 Reagent  according to the manufacturer's instructions. Cells were transfected with 0.5 μg of the indicated reporter plasmids, 0.5 μg of pSG5-hAR (where specified), and 0.5 μg of pcDNA3 or wild-type ebp1 expression plasmids and 5 ng of the TK-Renilla plasmid  as an internal control. Complete medium was replaced 24 h after transfection with phenol red free RPMI 1640 with CSS with or without R1881 (10−8 M) (Cells (5 × 10(10−8 M) . LuciferM final concentration of annealed oligonucledotides using Lipfectamine 2000 according to the manufacturer's instructions. The Ebp1 siRNA sequences corresponded to the coding regions beginning at nucleotides 476 and 995 (Genbank accession number U87954). The targets sequences were AAGCGACCAGGAUUAUAUUCU and AAGUGAGGUGGAAAGGCGUUU respectively. These sequences do not match any other human genomic sequences as determined by BLAST analysis using the NCBI Website. Scrambled oligonucleotides of these sequences were used as negative controls. The next day, cells were transfected with an expression construct for wild-type AR and the MMTV-luciferase and TK plasmids using Fugene-6.The siRNA oligonucleotides were purchased from Dharmacon Research Inc . COS-7 cells were cultured in 12-well plates until 60% confluent. Cells in 1 ml of antibiotic-free culture media were transfected with 60 nt-test. Significance was established at P⩽0.05.Results were analysed using a two-tailed Students2DS promoter in COS cells transfected with wild-type AR, and the PSA promoter in LNCaP cells that harbor a mutant AR . As expected, R1881 stimulated luciferase activity four- to five-fold in DU145 and PC-3 AR cells  with the ectopic expression of ebp1  3.5-fold increase in the luciferase activity of the MMTV promoter in the absence of androgen. No such stimulation was observed in cells lacking AR. R1881 stimulation of the reporter plasmid was decreased by inhibition of Ebp1 expression, but this change was not significant at the P⩽0.05 level  for 0, 15, 60 and 120 min and 24 h. Cell lysates were immunoprecipitated with antibody to ErbB3 as described. Ebp1 was found in ErbB3 immunoprecipitates of untreated cells  overnight. Cells were then treated with 20 ng ml−1 of HRG β1 for 1 h, a time when we could not detect Ebp1 in ErbB3 immunoprecipitates. Cell lysates were immunoprecipitated with the Ebp1 antibody. Western blot analysis of the immunoprecipitates indicated that HRG treatment enhanced the interaction of Ebp1 with AR  reduced AR luciferase activity 55%. Maximal inhibition of 90% was observed at 0.5 μg of the ebp1 plasmid. This was more than the 80% inhibition previously observed (−1), previously demonstrated to increase association of Ebp1 and AR, significantly (P⩽0.05) enhanced Ebp1-mediated repression at low (0.1 and 0.2 μg) amounts of the Ebp1 plasmid (HRG enhances Ebp1 transcriptional repression. We reasoned that if HRG could change the association of Ebp1 with AR, it could affect Ebp1 induced repression of AR transactivation. LNCaP cells were transiently transfected with the MMTV luciferase reporter plasmid and limiting amounts of an observed  and prob plasmid .We have previously established that Ebp1, a protein cloned in our laboratory via its interactions with the ErbB3 receptor, inhibits AR-mediated transcription and growth of the AR positive LNCaP cell line (2 luciferase reporter in COS cells and the PSA luciferase reporter in LNCaP cells (2 reporter was more potent in COS-7 than HeLa cells. This variability has been attributed to the complement of transcription factors and coregulators in different cell types. Therefore, we examined AR transactivation of MMTV-luc in two androgen-independent prostate cancer cell lines, PC3 and DU145, that had been transfected with wild-type AR. Ebp1 inhibited AR-regulated transcription in these androgen-independent cells. Similarly, Cyclin D1 inhibits AR transactivation across a wide variety of both prostate and nonprostate derived cell lines (ebp1 overexpression completely inhibited AR activity in DU145 and PC-3 AR transfected cells, ebp1 was unable to completely suppress the response to R1881 in LNCaP cells. This discrepancy may have been due to different expression levels of AR in the DU145 and PC-3 cells lines as compared to LNCaP cells, the fact the AR receptor is mutated in LNCaP cells, or to different transfection efficiencies of the ebp1 plasmid among the three cell lines.This study first demonstrated that Ebp1 inhibition of AR transactivation was neither promoter nor cell type specific. We had previously demonstrated that Ebp1 inhibits AR transactivation of the artificial AREStudies using siRNA demonstrated that inhibition of endogenous Ebp1 expression resulted in increased activity of an androgen regulated reporter construct in the absence of androgens. No such effect was observed in cells not expressing the AR. Thus, the increase in promoter activity in the absence of androgens was mediated via the AR. This finding suggests that Ebp1 may play a role in inhibition of AR signalling in the presence of no or extremely low levels of androgens. Ebp1 has the ability to bind DNA  and histin vivo.Although Ebp1 was shown to be associated with ErbB3 in breast cancer cells (in vivo. However, the efficiency of the Ebp1 : AR interaction was relatively low in the absence of HRG. HRG treatment of LNCaP cells for 1 h enhanced the association of Ebp1 with AR in LNCaP cells. The basis of the increased association of Ebp1 with AR after HRG treatment is not known. We have found that HRG increases phosphorylation of Ebp1 in breast cancer cells (We then determined the effects of HRG on Ebp1 function. HRG treatment resulted in dissociation of Ebp1 from the ErbB3 receptor in LNCaP cells. The present studies also demonstrate that HRG regulated the interaction of Ebp1 with the AR receptor. First, we demonstrated that endogenous Ebp1 associated with endogenous AR The fact that HRG enhances the binding of Ebp1 to AR suggests that in the absence of HRG, Ebp1 may not optimally affect AR function. Indeed, HRG potentiated the ability of limiting amounts of Ebp1 to inhibit AR promoter activity. HRG has been previously shown to inhibit growth of AR+ but not AR− prostate cancer cell lines (In summary, Ebp1 is a potent corepressor of AR with broad specificity. Ebp1 maintains its corepressor activity independent of cell type and promoter examined. Thus, Ebp1 joins a small but growing group of AR corepressors (


Only the patients with malignant or undeterminate lesions underwent surgery. Most lesions (86%) diagnosed as malignant by cytology or after surgery were positive for galectin-3. The majority of lesions (94%) classified as benign by cytology or after surgery was negative for galectin-3. The positive and negative predictive values were 83 and 95%, respectively. When focusing on the undeterminate lesions, the sensitivity and specificity were 75 and 90%, respectively, while the positive and negative predictive values were 82 and 87%, respectively. The specificity and the positive predictive value were higher (100%) when considering the percentage of stained cells. Altogether these results show that galectin-3 constitutes a useful marker in the diagnosis of thyroid lesions classified as undeterminate by conventional cytology.Fine-needle aspiration cytology, which is well established to be accurate for the diagnosis of thyroid cancer, may be inconclusive for the follicular thyroid neoplasms. As galectin-3 was suggested to be a marker of malignant thyrocytes, we investigated whether this protein might be helpful in the diagnosis of aspirates classified as undeterminate by cytology. After establishing an easy processing of aspirates for galectin-3 immunodetection, a series of aspirates categorised as benign ( Thyroid nodules are very frequent in the population and their diagnosis by fine-needle aspiration cytology has been well established to be highly accurate to discriminate malignant from benign lesions  for routine cytological diagnosis, while at least two smears were realised for subsequent galectin-3 immunocytochemistry.The prospective analysis concerned the three types of cytological samples, that is the benign, the malignant and the samples classified as undeterminate. The term undeterminate meant that no definitive cytological diagnosis was possible. During the period of investigation, more than 1500 fine-needle aspirations of thyroid nodules were performed. As expected, most of these samples were undoubtedly benign at cytological diagnosis. After having observed that galectin-3 was always negative in our first series of benign cases, we then did not systematically analysed all the benign lesions for galectin-3 but focused on lesions which although unambiguously benign were highly cellular.n=135) consisted of 63 benign, 17 malignant and 55 undeterminate lesions. In four cases of malignant lesions, the analysed material was lymph nodes instead of thyroid tissue as no thyroid nodule was seen by echography. The cytological diagnosis of these lesions was unambiguously papillary thyroid carcinoma.The samples subsequently analysed for galectin-3 (n=114).Indication for surgery was based only on clinical and MGG cytological findings without taking into account the results of galectin-3 immunodetection. Consequently, only the patients with lesions diagnosed as malignant or undeterminate by cytology were supposed to be referred to surgery. Among the 55 patients whose lesions were classified as undeterminate, 34 underwent surgery because of the clinical context. The remaining 21 patients were carefully followed-up or were not operated on by the time we sent this manuscript. Therefore, analysis of galectin-3 results was performed for the lesions diagnosed as benign or malignant by cytology and the subset of undeterminate lesions for which histological diagnosis was available . Samples were aspirated, put in CytoLyt then centrifuged on slide and tested for galectin-3. Samples were also spread on slides and analysed for galectin-3 after air-drying only.To define the optimal time between aspiration of samples and galectin-3 detection, we tested the human papillary thyroid carcinoma BCPAP cells. These cells were incubated in CytoLyt for different times, then centrifuged on slides and tested for galectin-3. They were also centrifuged on slides without prior incubation in CytoLyt, dried on air for different times and then tested for galectin-3.Cells on slides were fixed in acetone at 4°C for 10 min followed by 10% formalin at room temperature for 10 min. After blockage of nonspecific binding sites with 30% normal human AB serum, cells were incubated overnight at 4°C with anti-human galectin-3 NCL-GAL3 mouse monoclonal antibody  diluted 1 : 100. Cells were then incubated with EnVision-peroxydase detection kit as recommended by the manufacturer . Enzymatic activity was visualised with 3,3′-diaminobenzidine (Dako). Slides were then washed, counterstained with haematoxylin and mounted in aqueous medium. For each experiment, the human papillary thyroid carcinoma BCPAP cell line was used as positive control. For each sample, a negative control was realised, which consisted of substitution of the primary antibody by an isotype-matched mouse nonimmune IgG1. Positivity for galectin-3 was assessed by two independent investigators taking into account the percentage of cells exhibiting staining in the cytoplasm and/or at the plasma membrane. The positive cases were classified+when staining was observed in less than 20% cells, ++ for staining in 20–50% cells and +++ for staining in more than 50% cells.Sensitivity, specificity, positive and negative predictive values of galectin-3 immunodiagnosis were calculated as follows. Sensitivity was defined as the ratio of the number of carcinomas that were galectin-3 positive to the total number of carcinomas. Specificity was defined as the ratio of the number of benign lesions, which were galectin-3 negative to the total number of benign lesions. The positive predictive value was calculated as the ratio of the number of galectin-3 positive carcinomas to the total number of galectin-3 positive lesions. The negative predictive value was calculated as the ratio of the number of galectin-3 negative benign lesions to the total number of galectin-3 negative lesions.We found that detection of galectin-3 was possible for aspirated samples put in CytoLyt. This transport medium allowed detection of the protein for samples that have been in CytoLyt as long as 48 h. To examine whether the delay between fine-needle aspiration and galectin-3 immunodetection could be longer than 48 h, we tested the human papillary thyroid carcinoma BCPAP cell line for galectin-3 after incubation in CytoLyt for different times (4 h–7 days). It was clearly shown that galectin-3 labelling decreased with time to be almost absent after 7 days . Consequn=63), malignant (n=17) or undeterminate (n=34) was prospectively analysed for galectin-3. Typical examples of galectin-3 labelling using NCL-GAL3 mouse monoclonal antibody are shown in n=15), cytoplasmic staining was associated with plasma membrane staining. No nuclear staining was seen.A series of 114 fine-needle aspirates processed as reported above and categorised as benign , most lesions  were positive for galectin-3 , the majority of benign lesions (94%) was negative for galectin-3. Concerning the five positive lesions, the percentage of labelled cells was less than 50% in all samples except one.Taking into account all the lesions classified as benign either by cytology or after surgery  while the negative predictive value was similar (93%); however, the sensitivity was lower (79%).When focusing the analysis of galectin-3 results on undeterminate lesions, galectin-3 was detected in most malignant lesions and absent in the majority of benign lesions . The senSince the first large series of Most of the published studies concern retrospective analyses made on histological specimens or paraffin-embedded cell blocks. They do not evaluate the interest of galectin-3 as a marker of malignancy in the subset of lesions for which no diagnosis is possible by cytology. The present work was a prospective study and focused on samples classified as underterminate by cytology, which are the samples of concern.First of all, we optimised a galectin-3 immunocytochemical method for fine-needle aspiration thyroid samples. We show here that galectin-3 immunodetection is possible in routine medical practice especially as an office procedure. The processing of the specimens is easy as only air-dried smears are required. This procedure is shorter and less expensive than imunohistochemistry performed on paraffin-embedded cell blocks, which is the technique most often reported for fine-needle aspiration samples  at the cell surface has been previously reported  for the specificity and the negative predictive value. When focusing on undeterminate lesions, the specificity and the negative predictive value were also the highest values and were almost equal to 90% . The facThe absence of galectin-3 detection in thyroid carcinomas and the presence of galectin-3 in benign thyroid lesions have been previously reported by others (An interesting finding of the present study is that when galectin-3 labelling was present in benign cases, the percentage of galectin-3 positive cells was lower than in malignant cases. When considering samples to be positive if they contained more than 50% stained cells, the specificity and the positive predictive value were higher than without considering the percentage of stained cells . No majoConsidering the malignant cases negative for galectin-3, other potentially interesting markers such as HBME1 or cytokeratin 19 (In conclusion, galectin-3 immunodetection in fine-needle aspiration thyroid samples is easy and possible in routine medical practice. This marker is useful in the diagnosis of thyroid lesions classified as undeterminate by conventional cytology, especially if semiquantitative results, that is to say the percentage of positive cells, are taking into account. Galectin-3 allows one to identify the subset of undeterminate lesions with poor risk of malignancy and consequently the subgroup of patients who, if in accordance with the clinics, may be carefully followed-up instead of being referred to surgery.


Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy.Autographa californica GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested in vitro and in vivo for hepatocyte and macrophage gene transfer.Fusions were made of Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages.in vitro and in vivo with GP64/Sendai chimeric envelope proteins.We demonstrate reduced macrophage transduction  These pin vitro ,5. In coansduced -8. We haansduced . Althougansduced  it wouldin vitro [in vivo [Autographica californica multiple nuclear polyhedrosisvirus, GP64, is also able to efficiently pseudotype lentiviral vectors [in vivo gene transfer of lentiviral vectors pseudotyped with either VSVg or GP64 showed comparable transduction profiles in murine livers [in vivo.Lentiviral vectors are commonly pseudotyped with VSVg which generates stable virions capable of transducing a broad range of cells both in vitro  and in v[in vivo ,11. Howe vectors . GP64 ps vectors . A compae livers . Thus, ae livers ,15, pseuPlasmodium berghei circumsporozoite protein [The engineering of retroviral envelope proteins for retargeting represent a challenge as modifications to viral envelope proteins often results in a significant reduction in viral titers -18. GP64 protein , avidin  protein , and gp1 protein . Lentivi protein . We have protein .in vivo studies. We constructed a Sendai-GP64 fusion protein and investigated the tropism of lentiviral vectors pseudotyped with this envelope protein for hepatocytes and macrophages in vitro and in vivo.The Sendai Virus Fusion (SV-F) protein utilizes a hepatocyte specific receptor for viral entry . Both mu0, which is cleaved by a cellular protease to a F1 and F2 chain [The Sendai Virus Fusion protein (SV-F) is expressed as an inactive precursor protein FF2 chain ,30  directed against GP64 gave the expected band for the wild type GP64 pseudotyped lentiviral vectors Figure  lane 1, Lentiviral vectors expressing GFP from the PGK promoter were produced with wtGP64, Sendai-GP64, or Sendai-spGP64 viral envelope proteins. Viral titers were determined on both HeLa  and HepG2 (a liver hepatoma cell line) cells. Lentiviral vectors pseudotyped with GP64 can efficiently transduce both HeLa and HepG2 cells, while both Sendai-GP64 and Sendai-spGP64 pseudotyped lentiviral vectors were only able to transduce HepG2 cells. Thus, both chimeric envelope vectors displayed a higher affinity for HepG2 cells as compared to wtGP64 Table . Viral tin vivo use. Therefore, to increase viral titers, lentiviral vectors were produced with wtGP64 and either Sendai-GP64 or Sendai-spGP64 envelope proteins. The ratio of wtGP64 to Sendai-GP64 or Sendai-spGP64 plasmid used during virus production were optimised to yield lentiviral vectors that had high titers on HepG2 cells while retaining a reduced affinity for HeLa cells. The specificities of viruses produced with different ratio's of wild type GP64 and Sendai-GP64 are shown in table The titers of lentiviral vectors with either the Sendai-GP64 or Sendai-spGP64 envelope alone were too low for in vivo.We have previously shown that Kupffer cells (liver macrophages) are the predominant cell type transduced within the liver . Therefo8 HepG2 transducing units, of GP64 , Sendai-GP64/wtGP64 , or Sendai-spGP64/wtGP64  pseudotyped lentiviral vectors. One week following viral injections, the mice were sacrificed and tissues were fixed in vivo. Liver sections were prepared from the left and medial lobes and GFP expression was directly observed using fluorescence microscopy. In liver sections of mice injected with wtGP64 lentiviral vectors, the majority of transduced liver cells were nonparenchymal cells  in the amount of transduced nonparenchymal liver cells in mice injected with Sendai-GP64/wtGP64 lentiviral vectors as compared to wtGP64 Table . Sendai-Our observation that Sendai-GP64/wtGP64 pseudotyped lentiviral vectors have a reduced affinity for RAW macrophages in vitro is thus confirmed by the lower transduction of liver macrophages in vivo.To validate the microscopy data, PCR amplification specific for integrated lentiviral vectors was performed on genomic DNA isolated from the liver. In the Sendai-GP64/wtGP64 and Sendai-spGP64/wtGP64 injected animals a weaker band is observed in liver genomic DNA, validating the results from the counting of GFP expressing cells in liver sections  by western blot. This was surprising because the Sendai-GP64 construct contains the full length GP64 cDNA with exclusion of the signal peptide.Lentiviral vectors produced without a viral envelope protein lack the ability to bind and fuse to target cells and are not capable of gene transfer in HepG2 cells under our experimental conditions . Thus, a functional envelope protein must be incorporated in the Sendai-GP64 pseudotyped virions. The most likely explanation for this discrepancy is that different posttranslational processing of Sendai-GP64 results in disruption of epitopes that are recognised by the antibody on wild type GP64.Sendai-GP64 uses the Sendai virus signal peptide, when we constructed a Sendai-GP64 fusion protein that uses the GP64 signal peptide we were able to detect low levels of Sendai-spGP64 on western blot Figure  lane 3 sin vivo, Sendai-GP64/wtGP64 lentiviral vectors exhibited a significant reduction of gene transfer to Kupffer cells  for rs Table . Mice rels Table  and thisThe titers of Sendai-GP64 pseudotyped lentiviral vectors are low and must be improved to be suitable for practical use. Inserting the Sendai fragment into a different site of GP64 or using a directed evolution approach might result in new and improved hybrid viral envelopes.Hepatocyte specific gene transfer using GP64 pseudotyped Feline Immunodeficient Virus (FIV) derived lentiviral vectors has been described . This hein vivo gene transfer through manipulations to the GP64 envelope protein. Further improvements in GP64 fusion proteins allowing for higher levels of expression may eventually lead to hybrid envelope proteins with complete retargeting without the need to co-express wild type GP64 envelope protein.In this study we have shown that it is possible to redirect The Sendai Virus Fusion cDNA was kindly provided by Dr. Allen Portner  in a mammalian expression vector. The GP6MCSGP64for 5'GGTACCATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGCA-3'GP64midRev 5'-TAGATGCTGTTGTTGTAGC-3'.The resulting PCR product was ligated into the pCR2.1 TOPO vector and sequenced. The GP64 coding sequence containing the MCS was released by a KpnI and SacII digest and subcloned into a KpnI and SacII digest of the pCDNA 3.1 GP64 to create pCDNA3.1 MCSGP64. The Sendai Virus F2 coding sequence was amplified with the following primers ClaISVF2f 5'-ATCGATATGACAGCATATATCCAGAGATC-3'ClaISVF2r 5'-ATCGATTCTCGACTGGGGAGCACCGGCAT-3'The resulting product was verified by sequencing and cloned into the ClaI site of MCSGP64 to create pCDNA 3.1 Sendai spGP64.Concentrated viral supernatants of GP64, Sendai-GP64/wtGP64, Sendai spGP64, and Sendai spGP64/wtGP64 pseudotyped lentiviral vectors were run on 10% SDS-PAGE and blotted onto nitrocellulose using the Bio-Rad Miniprotean III system. An antibody directed against a C terminal epitope in the GP64 protein, AcV5 (eBioscience 14-6995), was used at a 1:1000 dilution. A 1:1000 dilution of Goat anti Mouse HRP (Bio-Rad 170-6516) was used as a secondary antibody on all blots. Detection of reactive bands on western blots was performed using the Lumi-Light western Blot Substrate (Roche 12 015 200 001) and blots were analyzed using a LumiImager F1 and LumiAnalyst 3.1 software (Roche).2. RAW mouse macrophages were grown in standard RPMI media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 10% CO2HEK293T, HeLa, and HepG2 cells were grown in standard DMEM media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 10% COLentiviral vectors with the phosphoglycerate kinase promoter driving eGFP expression were produced as described earlier . Briefly8 transducing units/ml and p24 levels ranged from 4-30 μg/ml.Viral particles were measured using a commercial ELISA kit for the Gag p24 protein (Perkin Elmer NEK050). HepG2 titers for the concentrated lentiviral vectors ranged from 1.5 - 3.2 × 10ad libitum on standard laboratory chow. All animal experiments were performed in accordance with the Animal Ethical Committee guidelines at the Academic Medical Center of Amsterdam.Wild-type FVB male mice ages 6-10 weeks were used in all studies and were fed 2O, 7 ml/kg). Under deep anesthesia, the peritoneal cavity was opened and the mice were injected intraportally with identical amounts of infectious virions, the equivalent of 0.5 × 108 HepG2 transducing units, on day 0. The amount of HIV p24 injected was 6.3 μg for GP64, (0.25 ml of 2.0*108 TU/ml n = 5), 21.3 μg for Sendai-GP64/wtGP64, (0.35 ml of 1.5*108TU/ml) n = 7), and 4.6 μg for Sendai-spGP64/wtGP64,  n = 4. The peritoneal cavity was sutured and the animals received the analgesic Temgesic  subcutaneously following recovering from FFM.Mice were anesthetized with an intraperitoneal injection of FFM mix , followed by 20 ml of 2% formaldehyde in PBS. Following perfusion, the liver and spleen were removed and further fixed for 4 hours in 4% formaldehyde in PBS at room temperature. The fixed tissues were then transferred to 30% sucrose solution and incubated overnight at 4°C, snap frozen in liquid nitrogen and stored at -80°C the following day.On day 7, the mice were killed by Cryosections were made from both the left and medial lobes. The tissue was embedded in Tissue-Tek OCT (Bayer) and sections (6 μm) were applied to poly lysine coated glass slides and enclosed in Vectashield mounting media (Vector Laboratories).Liver sections were prepared as described above and frozen without mounting media. Following thawing at room temperature sections were washed three times 5 minutes each in PBS and blocked with 10% Normal Goat Serum in PBS/0.05% Tween-20 for one hour. Kupffer cells were stained with a rat anti mouse F4/80 antigen (1:20 Serotec) for one hour. Slides were washed three times 5 minutes each in PBS/0.05% Tween-20 and incubated with a goat anti rat Texas Red conjugated antibody  for one hour. Following three washing steps of 5 minutes each, sections were embedded in Vectashield mounting media containing DAPI. Images were captured at (400×) magnification using a fluorescent microscope (Leica DMRA2).GFP positive cells were counted in sections made from the left and median lobes using a fluorescent microscope (Leica DMRA2). All sections/slides were prepared and coded independent of the counter. Identification of parenchymal and nonparenchymal cells was made based on morphology as described before .2 was estimated by counting amount of hepatocytes present in one field at (400×). Data are reported as number of GFP expressing cells per square millimeter.Per animal, one section from the left lobe and median lobe were counted for GFP positive hepatocytes at (200×). Three fields (200×) per section were counted for GFP positive nonparenchymal liver cells. Images of the counted sections were captured and the surface area of sections was calculated using Leica FW4000 software. The number of total hepatocytes per mmStatistical analysis was performed using SPSS 11.0 software using the Mann-Whitney U test. Values were determined to be significantly different with p < 0.05.Genomic DNA was isolated from snap frozen liver and spleen tissue using Dneasy tissue kit (Qiagen) according to manufacturers instructions. The following primer pairs were used to generate a 274 bp product: HIV-U3 forward primer 5'-CTGGAAGGGCTAATTCACTC-3' and HIV PSI reverse primer 5'-GGTTTCCCTTTCGCTTTCAG-3'. This primer pair is designed to specifically amplify integrated provirus and thus reduce contamination from amplification of the transfer plasmid. Additionally primers directed against GAPDH were used as a template loading control GAPDH forward primer 5'-CAATCACCATCTTCCAGGAG-3' and GAPDH reverse primer 5'-TGCCCACAGCCTTGGCAGC-3'. 100 ng of total DNA was used per PCR reaction using the following conditions: 95°C for 5 minutes, followed by 33 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds with a fill in at 72°C for 10 minutes. Negative control samples were taken from animals that had not been injected with virus.DMM and JS conceived the study.DMM, NPVT, JKH and JS designed and performed experiments.DMM, RPJOE and JS analyzed the data.DMM and JS wrote the manuscript.All authors read and approved the manuscript.


RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s).We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death.We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences. TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis in many transformed cell types but not in most normal cell types ,2. This TRAIL stimulates apoptosis by binding to one of two receptors, TNFRSF10A and TNFRSF10B ,5. This Investigations into the mechanisms that regulate sensitivity of cells to TRAIL have implicated many factors and pathways. Regulation of the TRAIL receptors, at the level of expression, localisation to the cell surface and the O-glycosylation of the proteins, partially, but not fully correlates with sensitivity -11. LeveGenome scale gene knock-down screens using RNA interference (RNAi) are an increasingly popular method for finding genes associated with cellular phenotypes. Indeed two screens for genes involved in TRAIL-induced apoptosis have been undertaken ,21. DespOff-target effects in RNAi screens occur where short interfering RNAs (siRNAs) directly affect the expression of genes other than the one that they are designed to target. Micro-array experiments have shown that an siRNA can affect the mRNA levels of many genes ,23. Theret al found that their top three hits scored well due to off-target effects. They observed that two of these three siRNAs shared the same 7 nt seed sequence. Searching a database of 3' UTRs, they found that the Bcl-2 family member Mcl-1 contained matches to both the 7 nt seed sequences seen in their top three siRNAs. They hypothesised that screening enriches for siRNAs with off-target effects and that this will lead to enrichment of certain seed sequence in high scoring siRNAs [Such off-target effects have been shown to cause problems in siRNA based screens. In a screen for genes which sensitize normally resistant cells to a Bcl-2 inhibitor, Lin g siRNAs .Until now, such effects in siRNA screens have only been described as post-hoc explanations for already identified off-target effects, generally restricted to the seeds of the top few siRNAs from a hit list. To the best of our knowledge, systematic analyses of the effects of seed sequences on siRNA screen results have yet to be reported. Therefore, here we report a screen for regulators of the TRAIL-induced apoptosis pathway. After rigorous confirmation of a set of hits, we investigated the off-target effects in our results. We show both that a set of seed sequences is over-represented in our potential hit siRNAs and that a second, overlapping set of seed sequences is enriched in high scoring siRNAs in general and that the presence of one of these seed sequences is sufficient to render an siRNA active in our screen. This effect was used to identify three species of miRNA that are regulators of the TRAIL induced apoptosis pathway.BID, CASP3, CASP8, DIABLO and TNFRSF10A, Figure In order to confirm that our assay is capable of identifying novel genes in the TRAIL-induced apoptosis pathway we selected 18 genes previously implicated in the pathway, including several genes identified in a previous screen, in addition to genes central to the apoptosis pathway . We measWe next screened a library of 12,190 siRNAs, targeting the 6,095 genes of the druggable genome, excluding kinase and phosphatase genes (which were screened separately), in duplicate, for novel genes in the TRAIL-induced cell death pathway , we were able to confirm that the effect of siRNAs targeting these genes was due to on-target effects by these criteria, therefore implicating these genes as genuine regulators of TRAIL-induced apoptosis. In the case of PDE11A we were unable to detect expression in any sample (possibly due to very low wild-type expression levels), but included the gene in our hit list since we found three siRNAs targeting PDE11A that have a significant effect on Caspase-3/7 levels. Kinase and phosphatase genes were investigated in a separate screen, but we were unable to confirm any hits (data not shown).In five cases , the opposite of the phenotype shown here [The function of product of own here . How thiDuring the confirmation of our screening results we encountered a large number of off-target effects - siRNAs which caused a phenotypic effect that was independent of their effect of the transcript level of the intended target. It is also worth noting that in several cases we identified genes where knock-down by two siRNAs induced TRAIL resistance, while a third siRNA did not, despite reducing the mRNA levels further, suggesting that the effect of both of the first two siRNAs were due to off-target effects  and that the off-target effects were due to matches to these sequences . To examet al's hypothesis that screening itself enriches for siRNAs with particular seed sequences. From here on we use 'enriched seed' to refer to both those seeds identified by GSEA and those that appear multiple times in the 16 confirmed siRNAs.Restricting our examination of seed sequences to the top 20 siRNAs is arbitrary. The Gene Set Enrichment Analysis (GSEA) algorithm tests for the enrichment of a set of items within a larger ranked list of such items . In ordeSince siRNAs with these seeds have a tendency to score highly, it is possible that they are scoring highly because they contain these seed-sequences rather than because the intended target is involved in TRAIL-induced apoptosis. To test if the presence of an enriched seed alone could alter the sensitivity of cells to TRAIL induced apoptosis, we transfected cells with either an siRNA targeting Luciferase (siGL2), or anti-luciferase siRNAs where the seed sequence (nucleotides 2-7) was replaced with either the sequence ACTTGA (siGL2+seed) or a version of the same sequence with two bases exchanged (ATCTGA - siGL2+mutseed). ACTTGA appears in the seed position of 3 of the 16 confirmed siRNAs, and was the second most enriched seed sequenced identified by GSEA. Cells transfected with the siRNA containing the mutated seed have a very similar sensitivity to TRAIL-induced cell death to that of cells transfected with the unaltered anti-Luciferase siRNA . In each of these cases we had found a second siRNA targeting the gene that also had a significant effect on TRAIL-induced apoptosis. However, in the case of TEGT that other siRNA (which was the second siRNA used in the screen) also contained one of the enriched seed sequences. Given the amount of evidence for the involvement of MYC in the TRAIL pathway from the literature, it is unlikely that siRNAs targeted against MYC exert their effect solely though off-target effects [In total 9 of the 16 siRNAs with a confirmed effect on Caspase-3/7 actiavation contain a seed from one of these two sets of sequences, including six of the top 7 siRNAs. This includes two siRNAs targeting genes that had been selected as confirmed hits  have one of the enriched seed sequences. In total the effects of the 12 of the 16 (75%) siRNAs which reproducibly reduced the sensitivity of cells to TRAIL-induced cell death could be attributed to either the targeting of a hit gene or the seed sequence of the siRNA. We cannot say that all the genes targeted by an siRNA containing a seed are definitely not involved in TRAIL-induced apoptosis, just that at least part of their effect is likely to be due to off-target effects. Even so the majority of siRNAs in the top 20 appear to owe at least part of their activity to off-target effects. Such findings suggest that screening enriches for siRNAs with off-target effects and that a high scoring siRNA in this assay is more likely to exert its effect through an off-target effect than by affecting the intended target. Thus off-target effects are probably more widespread than is generally recognised.TEGT in this study). We recommend that hits should be confirmed using at least two, and ideally three or more siRNAs not used in the screen, and whose seeds have been checked.The usual method for distinguishing true hits from RNAi screens from off-targets is to require that two independent siRNAs targeting the same gene have the same phenotype . HoweverFinally, we note that not only can seed sequences lead to a substantial number of false positives in RNAi screens, but can also mask true hits and thus increase false negative rates. In the TRAIL-killing screen described here, we tested for the enrichment of genes previously associated with the TRAIL pathway. We identified 27 genes that had previously been associated with the TRAIL induced apoptosis pathway and knockdown of which might be expected to decrease sensitivity to TRAIL-induced cell death. siRNAs against 12 of these were present in our library . We applied GSEA's pre-ranked analysis to test for enrichment of this gene set in the results from our screen. We found no significant trend, suggesting that we had failed to pick up most of the previous known genes involved in TRAIL killing  = 1.64, P = 0.097). To determine if this was due to a real effect being hidden by the off-target effects, we removed any siRNA containing an enriched seed sequence from our data and reanalysed the screening data. The ranking of siRNAs under this regime is available in Additional File Transfecting siRNAs to knock-down target genes is equivalent to over-expressing miRNAs of the same sequence as the siRNA. If many of the siRNAs that score well in our screen are exerting their effect through miRNA-like effects determined by a set of common seed sequences we reasoned that we might be able identify miRNAs with the same seed sequences and this might be an indication that these miRNAs are involved in regulating TRAIL-induced apoptosis. We searched a set of seed sequences found in human miRNAs downloaded from the miRBase database (version 10.0). We identified three miRNAs whose seed sequences were the same as those identified above Table .One of these miRNAs (miR-145) has previously been implicated in the regulation of TRAIL induced apoptosis . miRNA-2Caenorhabditis elegans miRNA used as a negative control. In two of the three cases (miR-26a and miR-145) this difference was still significant after correction for multiple testing. To test if the involvement of these miRNAs was specific to HeLa cells or extends to other cell types, we transfected DU145 (prostate carcinoma), HCT-116 and SW480  cells with the same miRNA mimics. The effects of the miRNA mimics were similar in SW480 and DU145 cells to HeLa cells, while in HCT-116 the effects were significant only in the case of the mimic of miR-126a  not used in the original screen, and do not contain seed sequences found in high scoring siRNAs from the screen.Where as previously off-target events have been regarded as unfortunate anomalies (which can in some circumstances be driven by miRNA-like targeting through seed sequences), our findings provides support for the hypothesis of Lin Removing siRNAs containing enriched seed sequences allowed us to show that siRNAs targeting genes known to be involved in the TRAIL induced apoptosis pathway were enriched in high-scoring siRNAs. This suggests that identifying enriched seed sequences and removing siRNAs containing them from analysis in the future will aid with such investigations.One obvious approach to combating such off-target effects would be to exclude siRNA with common seed sequences at the design stage . While this may exclude siRNAs with the largest number of off-target effects , seeds from endogenous miRNAs are not necessarily the ones that are most common in 3' UTRs. While siRNAs containing the seeds of endogenous miRNAs may have fewer targets, their targets are likely to have some degree of functional relation, and are therefore more likely to have a strong functional effect on the cell. Excluding siRNAs containing the top 10% of 6 mer seed sequence by frequency would have removed 6 of the 17 seed sequences we found to be enriched, but none of the seed sequences we identified that are found in endogenous miRNAs (data not shown). Excluding so many seed sequences also greatly reduces the design space for siRNAs and will result is more siRNAs sharing the same seed sequences .Finally we used the enriched seed sequences to identify miRNAs that may be involved in TRAIL-induced apoptosis, identifying miRNAs miR-26a, miR-145 and miR-384 as affecting TRAIL-induced cell death in a range of cell types. Further work will be needed to examine if these miRNAs have an endogenous role in regulating the TRAIL-induced apoptosis pathway. This analysis allows additional information to be gathered from the results of RNAi screening and turns potentially confounding off-target effects into a new source of information for the process being studied.miRNAs present an attractive candidate for controllers of sensitivity to TRAIL-induced apoptosis as they can control many genes simultaneously, allowing those changes necessary to transform cells to be linked to changes sensitizing them to TRAIL-induced apoptosis.HeLa cells were obtained from ATCC (#CCL-2) and were maintained in Modified Eagle's medium (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, 1× Non essential amino acids, penicillin and streptomycin. SW480, HCT116 and DU145 cells were a kind gift from Prof. Mike Stratton. DU145 cells were maintained in the same media as HeLa cells. SW480 cells were maintained in Leibovitz's L-15 Medium (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, penicillin and streptomycin. HCT116 cells were maintained in McCoy's 5a (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, penicillin and streptomycin.Individual siRNAs were obtained from either Invitrogen, Qiagen, Ambion or Dharmacon. Sequences and suppliers of siRNAs is listed in additional file Cells were transfected with 2.5 pmol siRNA in 96-well plates or 12.5 pmol siRNA in 24-well plates using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions for each cell type.For HeLa cells, 3,000 cells were seeded into the well of a 96 well optilux plate (BD Bioscience), in antibiotic free media. 24 hours later cells were transfected with siRNA. After a further 48 hours the number of cells present was measured by incubating for 3 hours with 10% alamarBlue reagent  before reading flourescence  using a fluorescent plate reader . Cells were treated with 0.5 μg/ml recombinant TRAIL  in serum-free media for 20 hours and number of cells present was reanalysed as above.BID and DIABLO as positive controls, two duplicates of an siRNA targeting siKIF11 as a transfection control, and four wells containing three different negative controls . The siRNAs were transfected into HeLa cells and the effect on TRAIL induced measured as described above. The screen was performed in duplicate. Plate were quality controlled by measuring the dynamic range between positive control transfected wells and negative control transfected wells, plates where the positive control was not twice the negative control were repeated.12,190 siRNAs from the Qiagen druggable genome library v2 were arrayed into 96-wells plates along with two duplicates of the siRNAs targeting Caspase-8, Data analysis was performed using the cellHTS package in R/Bioconductor . BrieflyThe minimum of the two scores for each siRNA was selected to represent that siRNA. See cellHTS report in supplementary information for analysis script.For each gene targeted by an siRNA in the top 20 scoring siRNAs, both siRNAs in the library were retested in triplicate. Where only one siRNA targeting a gene had a significant effect a further two siRNAs were obtained. siRNAs were tested for their effect on Caspase-3/7 activity. 3,000 cells were seeded in optilux plates and transfected with siRNA as described above. 48 hours after transfection cells were incubated with 0.5 μg/ml recombinant TRAIL in serum free media for 6 hours and the level of Caspase-3/7 measured using the Caspase-glo 3/7 kit (Promega) according to manufacturer's instructions.To measure the effect of siRNAs on mRNA levels, 15,000 cells were transfected with 12.5 pmol siRNA using 0.6 μl Lipofectamine 2000 and after 48 hours RNA prepared using the SV Total RNA isolated kit (Promega), and cDNA prepared using Superscript II (Invitrogen). SYBR green qPCR was performed using qPCR MasterMix (Eurogentec) with primers designed to amplify the transcript of interest or ACTB or GAPDH. All primers were tested to measure amplification efficiency. See additional file To test the significance of observing the same seed sequence in multiple siRNA from the top 20 siRNAs, the seed sequence of all siRNAs in the library were extracted. Samples of twenty seed sequences were selected at random, and the number of times seed sequences appeared counted. This was repeated 5,000 times for both hexamer and heptamer seed sequences and the number of times any seed sequence appeared more than one was counted.To use GSEA to detect enriched seed sequences, "gene-sets" were built containing the each of the siRNAs containing at particular seed sequence. The GSEA desktop application's pre-ranked analysis option was used to test if these sets were enriched in high-scoring siRNAs. This was repeated for both hexamer and heptamer seed sequences.GSEA: Gene Set Enrichment Analysis; miRNA: microRNA; RNAi: RNA interference; siRNA: small interfering RNA; TRAIL: TNF related apoptosis inducing ligand.IMS was involved in the conception, design and interpretation of the work, carried out all experimental work, screening data analysis, identification of seed sequences and wrote the manuscript. AJE was involved in the interpretation of the data, the analysis of the enriched seed sequences and editing the manuscript. AGF and ID were involved in the conception, design and interpretation of the work and editing the manuscript. The final manuscript has been reviewed and approved by all authors.Complete screen results (screen_results.zip). Complete results from the siRNA, presented as a mini-website as produced by the cellHTS softwareClick here for fileSupplementary figures (supplementary_figures.pdf). Figures S1 and S2 containing detailed results from confirmation experiments for all 16 genes targeted by siRNAs from the screen which reproducibly reduce sensitivity to TRAIL-induced cell death.Click here for fileRanking of siRNAs after removal of siRNAs with enriched seed sequences (no_enriched_seeds.csv). Ranking of siRNAs used in the screen according to their score, after any siRNA containing an enriched seed had been removed and data reanalysed.Click here for fileSequences of siRNAs used (siRNAs.csv). List of sequences, suppliers and catalogue numbers for non-library siRNAs.Click here for fileSequences of qRT-PCR primers (primers.csv). List of primer sequences used along with product lengths and amplification efficiency for each pair.Click here for file


The mechanisms and components that regulate macropinocytosis are poorly understood. Here we have investigated the role of sorting nexin 5 (SNX5) in the regulation of macropinocytic activity.P and PtdInsP2 through its PX domain, was recruited to regions on the plasma membrane containing EGF receptor or positive for PtdInsP2 as detected with the PH domain of TAPP1. Treatment with AG1478, an EGF receptor specific tyrosine kinase inhibitor, prevented the recruitment of SNX5 to the cytosolic face of the plasma membrane and inhibited the formation of macropinosomes in response to EGF treatment.SNX5 is abundantly expressed in macrophages, cells very active in macropinocytosis, and is recruited onto newly-formed macropinosomes. LPS treatment of bone marrow-derived macrophages resulted in a 2.5 fold decrease in macropinosome formation that correlates with a reduction in the levels of SNX5. To investigate the relationship between SNX5 levels and macropinocytic activity we examined the formation of macropinosomes in HEK-FlpIn cells stably expressing GFP-SNX5. Constitutive macropinocytosis was increased ~2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Furthermore, EGF stimulation resulted in a significant increase in macropinocytosis and there was also a 2.0 fold increase in the generation of macropinosomes in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. SNX5, which interacts specifically with PtdIns(3)Based on these data, we propose that SNX5 requires the generation of phosphoinositides for recruitment to the plasma membrane and, moreover, influences the level of macropinocytic activity. Macropinocytosis is an endocytic process that enables cells to internalize large amounts of solutes from the external environment. Macropinosomes are generated from the base of actin-mediated membrane ruffling when the lamellipodia folds back onto itself thereby forming very large endocytic structures. Macropinosomes are heterogeneous in size and generally considered to be > 0.2 μm in diameter ,2, a sizSalmonella and Shigella to gain entry into host cells [Macropinocytosis is important in a range of physiological processes. For example, macropinocytosis has a role in the down-regulation of signalling from the plasma membrane  and, becst cells .P2 [P2 [P3 [P, is implicated in the membrane trafficking of the endosomal system.Despite the physiological relevance of macropinocytosis, the molecular basis for the regulated formation and maturation of macropinosomes is very poorly understood. Macropinosome formation in a range of cell types has been shown to be phosphoinositide-3 kinase dependent  and unliP2 , PtdInsP2 [Sorting nexins are a large family of proteins characterised by the presence of a phox (PX) domain at the amino terminus. The modestly conserved PX domain is a sequence of 70 to 120 residues that has been shown to bind to various phosphoinositides hence the PX domain confers phosphoinositide specificity to the protein . SortingsP2  and SNX5sP2 . After EsP2 , charactsP2 . Our presP2 . Here, w® 488, goat anti-mouse IgG Alexa Fluor® 568, goat anti-mouse IgG Alexa Fluor® 647, biotinylated EGF complexed to Alexa Fluor® 555 streptavidin and Texas-Red-X Phalloidin were purchased from Molecular Probes . Horse-radish peroxidase conjugated rabbit anti-mouse and swine anti-rabbit immunoglobulins were from Dako . Full length human EGF-R was cloned into the XbaI sites of pCI-neo. The PH domain of TAPP1 (aa 95–400) was from Dr. S. Dowler .Rabbit anti-mouse SNX5 was raised against a synthetic peptide corresponding to the N-terminal 35 residues of mouse SNX5, with an additional Cys on the C-terminus, and affinity purified on a column of peptide conjugated to SulfoLink Coupling Gel . Monoclonal mouse antibodies to human EEA1 were from BD Transduction Laboratories . Mouse monoclonal anti-human EGF receptor (EGF-R) antibody (clone 528) has been described . MonocloHEK-FlpIn  and HEK-GFP-SNX5 stable cells were generated, cultured and transfected as previously described . PrimaryCell monolayers were cultured for various time periods in the presence of 100 μg/ml dextran  conjugated to tetramethylrhodamine (TR-dextran) . The cells were then washed with media at 4°C and fixed in 4% PFA. Images were captured using an LSM 510 Meta confocal microscope and quantified for macropinosomes using an automated image analysis pipeline in ImageJ 1.37v (NIH). The 'Substract Background' functionality was executed, and images thresholded to a binary image. Dextran-positive structures at least 0.5 μm in diameter were quantified through the 'Analyze Particles' feature.For analysis of macropinosomes using endosomal markers, cell monolayers were washed twice using PBS, serum-starved for 16–24 h at 37°C and stimulated with EGF (100 ng/ml)  at 37°C for 10 min. Cells were then washed twice in PBS, fixed in 4% PFA and stained using mouse anti-human EEA1 followed by goat anti-mouse Alexa Fluor 568. Macropinosomes were identified based on their size (>1 μm) [HEK-GFP-SNX5 cells, transiently transfected with pCI-neo-EGF-R for 24 h, were serum-starved for 16–24 h at 37°C. Cells were washed twice using ice-cold PBS and incubated with 100 ng/ml Alexa Fluor 555 conjugated-EGF  in serum-free media for 10 min on ice. Excess EGF was removed by washing the cells three times in ice-cold PBS. Cells were resuspended in ice-cold serum-free media (in the absence and presence of drugs) and the cells were then incubated at 37°C for the indicated duration to allow EGF internalization. Cells were then fixed and processed for immunofluorescence analysis.Cell monolayers were washed twice with PBS and serum-starved for 16–24 h at 37°C. Cells were treated with 100 nM AG1478  (from a 10 mM stock in DMSO) for 15 min at 37°C. EGF (100 ng/ml)  was then added to the cells in the presence or absence of AG1478 at 37°C for the indicated durations. Cells were then washed twice with PBS and processed for immunofluorescence.Cell monolayers grown on poly-L-lysine coated coverslips were washed twice in PBS and fixed in 4% PFA for 15 min. Cells were processed for immunofluorescence as previously described  and examLate endosomes and lysosomes of cells were labelled with fluorescently-conjugated dextran as described previously . BrieflyProteins were resolved on 10% SDS-polyacrylamide gels and transferred onto Immobilon-P PVDF membranes  according to the manufacturer's instructions and immunoblotting performed as described . SNX5 wat-test, two-tailed.Data were analysed by an unpaired Student's We have previously shown that GFP-SNX5 is associated with macropinosomes in HEK-FlpIn cells . The queTo directly explore the role of SNX5 in macropinocytosis we have used HEK-FlpIn cells which stably express SNX5-GFP. This cell line was the previously described HEK-FlpIn cell line transfected with pcDNA5/FRT-EGFPSNX5, referred to as HEK-GFP-SNX5 . HEK-GFPNext we investigated if the elevated level of SNX5 in HEK-GFP-SNX5 cells modulated the level of macropinocytosis. To estimate changes in macropinocytosis, newly-formed macropinosomes were labelled using a 5 min pulse of the fluid phase marker dextran . Only dextran-positive-structures >0.5 μm in diameter were scored as macropinosomes, and this size-based exclusion will minimise any contribution from changes in pinocytosis kinetics. Untreated serum-starved HEK-GFP-SNX5 cells showed a 1.6 fold increase (p < 0.02) in constitutive macropinocytosis compared with parental HEK-FlpIn cells Fig . PreviouWe then determined the impact of the elevated level of SNX5 on macropinocytosis after EGF stimulation. After a 5 minute EGF stimulation of serum-starved cells, HEK-GFP-SNX5 cells exhibited a 2.0 fold increase in the number of macropinosomes compared to HEK-FlpIn cells (p < 0.005), utilizing the Dextran-uptake assay Fig . These dNext we investigated the relationship between EGF-R signalling and macropinocytosis in the stable HEK-GFP-SNX5 cells using AG1478, an EGF-R specific tyrosine kinase inhibitor that prevents EGF-R activation . SignifiTo determine if the elevated macropinocytic activity induced by increased levels of SNX5 in HEK-GFP-SNX5 cells can support the internalization of EGF-R, we transfected HEK-GFP-SNX5 cells with EGF-R to increase the numbers of receptors for detection by immunofluorescence. Transfected HEK-GFP-SNX5 cells were incubated with Alexa555-conjugated EGF on ice, then the cells washed and incubated at 37°C to allow internalization of surface bound ligand-receptor complexes. As previously reported , SNX5 anUpon EGF-R activation, SNX5 is recruited to the plasma membrane and the newly-formed macropinosomes  and is cNext we investigated if SNX5 recruited to the plasma membrane after EGF stimulation can influence the rate of formation of macropinosomes. To determine the spatial relationship between plasma membrane recruitment and the location of EGF-R, serum-starved HEK-GFP-SNX5 cells were treated with EGF for 3 min and non-permeabilised cells stained with anti-EGF-R antibodies. Although the staining intensity of the endogenous cell surface EGF-R was weak, the receptor was detected in the ruffling edges of the cell and showed a co-localization with GFP-SNX5 (not shown). To enhance the sensitivity, this experiment was repeated with transfected HEK-GFP-SNX5 cells expressing elevated levels of EGF-R Fig. . GFP-SNXP3 and its breakdown product, PtdInsP2. We have shown that the PX domain of SNX5 binds to both PtdIns(3)P as well as PtdInsP2. To determine the relationship of PtdInsP2 generated from activated EGF-R and the plasma membrane recruitment of SNX5, we have used the PtdInsP2-specific probe, mCherry-TAPP1-PH domain [P2 at the plasma membrane. HEK-GFP-SNX5 cells were transfected with mCherry-TAPP1-PH domain, then the cells serum-starved and treated with EGF for 5 min. mCherry-TAPP1-PH was located on the plasma membrane of EGF-stimulated cells, but not non-stimulated cells, and showed co-localization with GFP-SNX5, demonstrating that SNX5 was recruited to regions of the plasma membrane rich in PtdInsP2 after EGF stimulation H domain , to deteion Fig. . Signifiion Fig. . Qualitaion Fig. . The resThe mechanisms that drive macropinocytosis are poorly defined. Our previous studies showed that SNX5 associates with newly-formed macropinosomes following EGF stimulation . Given tP and PtdInsP2. [P and PtdInsP2, also based on liposome binding assays [P2. Cell signalling is essential for the recruitment of SNX5 to the cytosolic face of the plasma membrane, as AG1478 inhibited the movement of SNX5 to the plasma membrane, presumably due to the lack of PtdInsP3 and its breakdown product PtdInsP2.. However, AG1478 does not prevent EGF-R internalization via the clathrin-dependent pathway (unpublished observations), indicating, at least in this system that receptor dimerization is sufficient for clathrin-mediated endocytosis, and consistent with previous findings [The PX domain of SNX5 has the capacity to bind both PtdIns(3)P2. . The PI g assays . The plafindings .Given the association of EGF signalling and the behaviour of SNX5, it is formally possible that SNX5 is modulating the EGF-R signalling cascade and thereby influencing macropinocytosis indirectly. However, our data argues for a direct role of SNX5 in enhancing the early events of macropinocytosis rather than potentiating EGF-R signalling. Firstly, constitutive macropinocytosis in the absence of EGF stimulation is increased >2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Secondly, the kinetics of macropinosome maturation and the rate of EGF-R degradation is similar in parental and HEK-GFP-SNX5 cells following EGF stimulation, indicating SNX5 is promoting the early events driving the biogenesis of macropinosomes, but does not influence the rate of maturation and delivery to lysosomes.in vitro and in vivo. As SNX5 has a coiled-coil C-terminal and a predicted BAR domain, it is likely that it exists as a dimer. We have previously demonstrated that SNX5 does not form a homodimer but can associate with SNX1 and that association of SNX5 with endosomes appears to be dependent on the formation of SNX5/SNX1 heterodimers [A number of the SNXs can form homodimers and heterodimers rodimers . SNX5 isrodimers . TherefoP arising from the hydrolysis of PtdInsP2. The BAR domain of SNX5, in a complex with other sorting nexins, has the potential to distort the limiting membrane of the macropinosome into extensive tubular elements that subsequently separate from the macropinosome and traffic in a microtubule-dependent manner to perinuclear endosomes. As the tubular structures depart from the macropinosome there is a reduction in the surface area of the macropinosome, which matures into a Rab7-positive structure and eventually fuses with late endosomes/lysosomes. The identification of machinery that regulates this process now provides the ability to directly assess the biological role of macropinocytosis in a range of physiological settings.Based on our published data ,25 and tM-CSF: macrophage colony-stimulating factor; EGF: epidermal growth factor; SNX5: sorting nexin 5; TR-dextran: dextran conjugated to tetramethylrhodamine; GFP: green fluorescent protein; BMMs: bone marrow-derived macrophages; LPS: lipopolysaccharide; HEK-GFP-SNX5: HEK293 cells stably expressing SNX5-GFP; PtdIns: phosphoinositides; PX: phox domain; PFA: paraformaldehydeJPL generated the anti-mouse SNX5 antibodies and carried out the macropinosome analysis and inhibition assays. JTHW performed the dextran uptake and the computational quantitation of macropinosome formation. MCK carried out the macrophage analyses and macropinosome maturation assays. RDT participated in the design and co-ordination of the study and helped to draft the paper. PAG participated in the design and co-ordination of the study and drafted the paper. All authors read and approved the final manuscript.