Extended Data Fig. 10: Further characterization of the effect of stretch depending on the existence of FBs.
a. Tensin1-GFP transfected cell before (cyan) and during (magenta) stretch. Images aligned at an adhesion located centrally under the nucleus. Scale bar is 25 µm / 5 µm zoomed. b. Analysis of the whole cell strain and the strain under the nucleus upon a 10% stretch. Two-tailed paired t-test from 3 independent experiments (n = 14 cells). c,d. Cell membrane before and during (dashed line) stretch (c) and quantification of the percentage change of cell area (d) for cells cultured in scrambled or PUR4 peptide. Scale bar 20 µm. (Scramble n = 47, PUR4 n = 51 cells from 3 independent experiments; Two-tailed Mann-Whitney test). e,f. Hoechst-stained nuclei before and during (dashed line) stretch (e) and percentage change in nuclear area upon stretch (f) for cells on control and gluteraldehyde blocked substrates. Scale bar 5 µm. (Control n = 41, cells glut n = 35 cells, from 3 independent experiments; Two-tailed Mann-Whitney test). g,h. Cell membrane before and during (dashed line) stretch (g) and quantification of the percentage change of cell area (h) for cells on control and gluteraldehyde blocked substrates. Scale bar 20 µm. (control n = 41 cells, glut n = 35 cells from 3 independent experiments; Two-tailed Mann-Whitney test). i,j. Cell membrane before and during (dashed line) stretch (i) and quantification of the percentage change in cell area (j) in cells expressing GFP-only or Plectin 1f-8-GFP. Scale bar 20 µm. (GFP n = 46 cells, Plectin 1f-8 n = 51 cells from 3 independent experiments; Two-tailed Mann-Whitney test). k,l. Vimentin-GFP transfected cells before (cyan) and during (magenta) mechanical stretch (k) and quantification of percentage change in vimentin area upon stretch for scramble and PUR4 peptide (l). Scale bar is 20 µm. (Scramble n = 50 cells, PUR4 n = 47 cells from 3 independent experiments. Two-tailed Mann-Whitney t-test). m,n. γ-H2AX staining (m) and quantification of γ-H2AX intensity (n) in cells lacking cell-cell contacts that were not stretched (NS) or stretched (S) in presence of scramble or PUR4 peptide. Scale bar is 20 µm. (2-way ANOVA from 2 independent experiments, scramble NS n = 43 cells, scramble S n = 51 cells, PUR4 NS n = 43 cells, PUR4 S n = 49 cells). o,p. γH2Ax staining (o) and quantification of γ-H2AX intensity (p) in cells on control or glut blocked substrates that were not stretched (NS) or stretched (S). Scale bar 20 µm. (Control NS n = 156, control S n = 138, glut NS n = 196, glut S n = 140 nuclei from 2 independent experiments; 2-way ANOVA with Tukey’s multiple comparison test). Data are presented as mean +/- SEM.
