Fig. 4.

Clathrin-coated plaques regulate stiffness-dependent Erk signaling. a Western-blot analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated glass and treated with control or β5-specific siRNAs as indicated (representative image of four independent experiments). Total-Erk was used as a loading control. b Densitometry analysis of bands obtained in western-blots as in a. Results are expressed as mean ± SD from four independent experiments (*P < 0.05, one-way analysis of variance—ANOVA). c Western-blot analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated, 0.1 kPa polyacrylamide gels and treated with control or integrin β5-specific siRNAs, as indicated. Total-Erk was used as a loading control (representative image of three independent experiments) d Densitometry analysis of bands obtained in western-blots as in c. Results are expressed as mean ± SD from three independent experiments. e GFP-Erk-expressing HeLa cells treated or not with a β5-specific siRNA and transfected or not with TfR-mCherry, as indicated, were seeded on anti-mCherry antibodies-coated glass and imaged 24 h later. Scale bar: 10 µm. A color-coded scale for low and high signal intensity is shown. f Quantification of GFP-Erk nuclear enrichment index in cells as in e and cultured on control antibody- or anti-mCherry antibody-coated glass, as indicated, and treated or not with indicated siRNAs. Results are expressed as mean ± SD (*P < 0.01, **P < 0.001, ANOVA. ControlAb-siCtr: 114 cells from n = 5 independent experiments; mChAb-SiCtrl: 119 cells from n = 5 independent experiments; ControAb-siβ5-1: 146 cells from n = 5 independent experiments; mChAb-Siβ5-1: 120 cells from n = 5 independent experiments; ControlAb-siβ5-1 + siAP-2: 69 cells from n = 3 independent experiments; mChAb-siβ5-1 + siAP-2: 68 cells from n = 3 independent experiments; ControlAb-siβ5-1 + siCHC: 63 cells from n = 3 independent experiments; mChAb-siβ5-1 + siCHC: 67 cells from n = 3 independent experiments)