|
{"SUPP CAPTION FIGS6-1.png": "'\\n\\n**Supplementary Figure 6. Reservoirs and VLDs are unrelated to caveolae**. **a**, Time sequence of pEYFP-mem and Cav1-mcherry transfected cells before, during, and after application of 6% stretch for three minutes. Image to the right shows the lack of co-localization between pEYFP-mem (green) and Cav1-mcherry (red). **b**, pEYFP-mem transfected cell (green in merged image) fixed right after restoring iso-osmotic media and stained for caveolin 1 (red in merged image). No co-localization was observed between caveolin and VLDs. **c**, Time sequence of pEYFP-mem and'", "SUPP CAPTION FIGS1.png": "'\\n\\n**Supplementary Figure 1. Stretch system.****a**, Schematic of stretch system. A ring clamping a PDMS membrane (shown in red) is placed on a support containing a central loading circular post and an external ring, with an opening in between. Imaging objectives can then be placed either below (inverted microscope) or above (upright microscope). Cells are then cultured on the membrane after coating with fibronectin. **b**, Once vacuum is applied through the opening, it deforms and stretches the membrane. In some cases, cells were seeded on polyacrylamide and soft silicone gels previously attached to the membrane (see methods). **c**, Level of strain in the X and Y axis obtained after applying different vacuum suction pressures. Stretch was biaxial.\\n\\n'", "SUPP CAPTION FIGS2.png": "'\\n\\n**Supplementary Figure 2. Response of cells to 2% strain.** Response of a peYFP-mem transfected cell to the application of 2% strain for three minutes. No visible effects were observed either upon stretch application or upon stretch release. Insets display magnifications of the areas in red, showing that membrane ruffles were not flattened upon stretch application, and reservoirs were not formed upon stretch release. Scale bar is 20 mm.\\n\\n'", "SUPP CAPTION FIGS9.png": "'\\n\\n**Supplementary Figure 9. Adhesion dependence of reservoirs and VLDs.****a**, Time sequence of pEYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells and cells treated with 10 mg mL-1a5b1 antibody. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) (n=100/150 reservoirs from 10/6 cells). **c**, Corresponding quantification of mean reservoir diameter (n=250/200 reservoirs from 8/6 cells). **d**, Corresponding quantification of mean reservoir density (n=30/60 regions from 5/5 cells). **e**, Time sequence of pEYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells and cells treated with 10 mg mL-1a5b1 antibody. **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/40 VLDs from 10/5 cells. **g**, Corresponding quantification of mean VLD diameter (n=100/150 VLDs from 10/7 cells). **h**, Corresponding quantification of mean VLD density (n=50/70 regions from 8/7 cells). Scale bars indicate 20 mm. N.s., non-significant, *, p<0.05, ***, p<0.001. Scale bars are 20 mm. In all cases, Zoomed insets (10x10 mm) show the formation and evolution of membrane structures.\\n\\n'", "SUPP CAPTION FIGS10.png": "'\\n\\n**Supplementary Figure 10.** Model predictions. **a**, Model prediction for reservoir length upon stretch release as a function of applied stretch. **b**, model prediction for VLD diameters formed after restoring iso-osmotic medium from osmotic shocks of different magnitude. In a and b, red line corresponds to the bending modulus used throughout the work, dashed black line shows the prediction for a 5-fold increase in bending modulus. **c**, VLD shape after restoring osmolarity from hypo-osmotic shocks of different magnitude and **d**, VLD shape after restoring osmolarity from a 50% hypo-osmotic shock in a pre-stretched membrane (top) and then de-stretching the membrane by 6% (top).\\n\\n'", "SUPP CAPTION FIGS5.png": "'\\n\\n**Supplementary Figure 5. Poroelasticity of polyacrylamide gels.****a**, graph showing the height of polyacrylamide gels during and after application of 6% stretch for three minutes. Values are shown normalized to the height before stretch application. As gels are stretched, they first decrease their height to maintain volume, but they progressively incorporate water and swell. Once stretch is released, the progressive reduction in thickness is indicative of water expulsion flow. **b**, As a control, gels submitted to a 50% decrease in media osmolarity did not modify their height. (n=5 gels)'", "SUPP CAPTION FIGS7.png": "'\\n\\n**Supplementary Text:**_Abb_ and **inversion** (**Supplementary Text:**_Abb_, **Abb'", "SUPP CAPTION FIGS6.png": "'\\n## References\\n\\n* [1] A. B.\\n\\n'", "SUPP CAPTION FIGS3.png": "'\\n\\n**Supplementary Figure 3. Dynamic formation and resorption of membrane structures.****a,** Quantification of VLD fluorescence after re-application of iso-osmotic medium (1:maximum fluorescence, 0: background). n= 20 VLDs from 2 cells. Imaging was carried out using an inverted microscope (60x objective) which allowed to visualize the initial VLD formation period. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background). n= 100/80/30 reservoirs from 10/3/3 cells. Imaging was carried out using an upright microscope (60x objective). Both decreasing temperature (pink symbols) and increasing stretch magnitude (black symbols) slowed the process of reservoir formation. Images to the right show corresponding examples of membrane structures at the times indicated in the graph. **c**, Quantification of VLD fluorescence after re-application of iso-osmotic medium (1: initial fluorescence, 0: background). n= 100/50/60 VLDs from 10/5/4 cells. Imaging was carried out using an upright microscope (60x objective). Both decreasing temperature (pink symbols) and increasing the magnitude of the hyposomic shock (black symbols) slowed the process of VLD formation. Images to the right show corresponding examples of membrane structures at the times indicated in the graph. All insets have a size of 10x10 um.\\n\\n'", "SUPP CAPTION FIGS7-2.png": "'(n=100/100 reservoirs from 10/4 cells). **c**, Corresponding quantification of mean reservoir diameter (n=250/100 reservoirs from 8/4 cells). No significant differences were observed. **d**, Corresponding quantification of mean reservoir density (n=30/30 regions from 5 cells). **e**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells and cells treated with 0.5 mM cytochalasin D. Zoomed insets show the formation and evolution of membrane VLDs. **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/100 VLDs from 10/5 cells. **g**, Corresponding quantification of mean VLD diameter (n=100/60 VLDs from 10/3 cells). **h**, Corresponding quantification of mean VLD density (n=50/30 regions from 8/3 cells). **i**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells at 37(r) and cells at 26(r). Zoomed insets show the formation and evolution of membrane reservoirs. **j**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) n=100/60 reservoirs from 10/3 cells. **k**, Corresponding quantification of mean reservoir diameter (n=250/140 reservoirs from 8/3 cells). No significant differences were observed. **l**, Corresponding quantification of mean reservoir density (n=30/30 zones from 5/3cells). No significant differences were observed. **m**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes for control cells at 37(r) and cells at 26(r). Zoomed insets show the formation and evolution of membrane VLDs. **n**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=100/24 VLDs from 10/3 cells. **o**, Corresponding quantification of mean VLD diameter (n=100/45 VLDs from 10/3 cells ). No significant differences were observed. **p**, Corresponding quantification of mean VLD density (n=50/25 zones from 8/3 cells). No significant differences were observed. Scale bars indicate 20 mm. N.s., non-significant, *, p<0.05, ***, p<0.001. Scale bars are 20 mm.\\n\\n'", "SUPP CAPTION FIGS6-2.png": "'Cav1-mcherry transfected cells before, during, and after application of 50% hypo-osmotic media for three minutes. Image to the right shows the lack of co-localization between peYFP-mem (green) and Cav1-mcherry (red). **d**, peYFP-mem transfected cell (green in merged image) fixed right after releasing 6% stretch and stained for caveolin 1 (red in merged image). No co-localization was observed between caveolin and reservoirs. **e**, Time sequence of peYFP-mem transfected cells before, during, and after application of 50% hypo-osmotic media during three minutes. Zoomed insets show the formation and evolution of VLDs. Caveolin **1** knock-out cells were reconstituted either with caveolin 1-GFP or an empty control vector (IRES-GFP). **f**, Quantification of VLD fluorescence after re-application of iso-osmotic media (1: initial fluorescence, 0: background). n=50/25 VLDs from 5/3 cells. **g**-**h**, Corresponding quantification of mean VLD diameter (**g**) (n=100/60 VLDs from 5/3 cells) and density (**h**) (n=30/20 regions from 5/3 cells).No significant differences were observed. **i**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes. Zoomed insets show the formation and evolution of membrane reservoirs. Caveolin **1** knock-out cells were reconstituted either with caveolin 1-GFP or an empty control vector (IRES-GFP). **j**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background) (n=30/60 reservoirs from 3/4 cells) . **k**, Corresponding quantification of mean reservoir diameter (n=250/100 reservoirs from 3/3 cells). No significant differences were observed. **l**, Corresponding quantification of mean reservoir density (n=40/20 regions from 3/3 cells). No significant differences were observed. **S**cale bars are 20\\\\(\\\\mu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS7-1.png": "'\\n\\n**Supplementary Figure 7. Actin and temperature dependence of reservoirs and VLDs.****a**, Time sequence of peYFP-mem transfected cells before, during, and after application of constant 6% stretch during three minutes for control cells and cells treated with 0.5 mM cytochalasin D. Zoomed insets show the formation and evolution of membrane reservoirs. **b**, Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background)'", "SUPP CAPTION FIGS4.png": "'\\n\\n**Supplementary Figure 4. Co-localization of actin and membrane structures before, during and after stretch and hypo-osmotic shocks.****a**, cells transfected with pEYFP-mem and Lifeact-Ruby before, during, and after application of 6% stretch for 3 minutes. **b**, equivalent images obtained before, during, and after application of a 50% hypo-osmotic shock for 3 minutes. Insets (10x10 \\\\(\\\\upmu\\\\)m) show zoomed views of membrane and actin structures. Scale bars are 20\\\\(\\\\upmu\\\\)m.\\n\\n'", "SUPP CAPTION FIGS8.png": "'\\n\\n**Supplementary Figure 8. Membrane reservoirs and VLDs are observed across cell types and species.** Images of Chinese Hamster Ovary (CHO) cells, A431 human squamous carcinoma cells, and human keratinocytes (HaCaT) transfected with pEYFP-mem before and after being submitted to biaxial stretch (a) or hypo-osmotic shock (b). All cell types consistently showed the formation of reservoirs or VLDs, respectively. Zoomed insets (10x10 \\\\(\\\\upmu\\\\)m) show a magnification of both membrane structures. Scale bar indicates 20 \\\\(\\\\upmu\\\\)m.\\n\\n'"} |
