Figure 2.
ppMyosin area and traction force are dependent on β3 integrin and ICAP-1 expression. (A) β1 integrin KO osteoblast cell lines were treated with β3 integrin siRNA or scramble siRNA for 48 h and then let spread on FN coated glass for 24 h. Staining of myosin phosphorylation (ppMLC antibody, red) and β3 integrin (LucA.5, green) was performed and analyzed by fluorescent confocal microscopy. Silencing the expression of β3 integrin leads to the complete abolishment of the ppMLC-decorated stress fibers in the β1−/−/icap-1−/− and shrinkage of the cell area. Scale bar, 20 µm. (B) Quantification of the ppMLC area normalized to the cellular surface area after treatment with β3 integrin siRNA (si β3, clear colors) or with scramble siRNA (si Scr, dark colors). Customized particle analysis script from ImageJ was used after application of Unsharpen mask and Despecle filters. The error bars represent SD. N ≥ 42 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (C and D) Representative traction forces maps (TFM) from β1 integrin+/+-icap-1−/− and β1 integrin−/−-icap-1−/− cell lines treated with β3 integrin siRNA (siβ3, bottom panels) or with scramble siRNA (si Scr, upper panels) and quantification (D) of the total force applied (nN) on fibronectin-coated polyacrylamide gel with a defined rigidity of 5 kPa. The additional silencing of β3 integrins in the β1−/−/icap-1−/− cells decimates the traction forces, confirming that, in absence of β1 integrin and ICAP-1, generation of strong cellular contractility at adhesion sites is dependent on β3 integrins. Scale bar, 10 μm. Error bars represent SD. N = 80 cells. ****, P value ≤ 0.0001. (E) FACS analysis of median fluorescence intensity of β3 integrin on cell surface in β1 integrin KO osteoblast cell lines. Error bars represent SD. (F) Western blot showing the efficiency of β3 integrin silencing in β1 integrin+/+-icap-1−/− and β1 integrin−/−-icap-1−/− cell lines. Source data are available for this figure: SourceData F2.

ppMyosin area and traction force are dependent on β3 integrin and ICAP-1 expression. (A) β1 integrin KO osteoblast cell lines were treated with β3 integrin siRNA or scramble siRNA for 48 h and then let spread on FN coated glass for 24 h. Staining of myosin phosphorylation (ppMLC antibody, red) and β3 integrin (LucA.5, green) was performed and analyzed by fluorescent confocal microscopy. Silencing the expression of β3 integrin leads to the complete abolishment of the ppMLC-decorated stress fibers in the β1−/−/icap-1−/− and shrinkage of the cell area. Scale bar, 20 µm. (B) Quantification of the ppMLC area normalized to the cellular surface area after treatment with β3 integrin siRNA (si β3, clear colors) or with scramble siRNA (si Scr, dark colors). Customized particle analysis script from ImageJ was used after application of Unsharpen mask and Despecle filters. The error bars represent SD. N ≥ 42 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (C and D) Representative traction forces maps (TFM) from β1 integrin+/+-icap-1−/− and β1 integrin−/−-icap-1−/− cell lines treated with β3 integrin siRNA (siβ3, bottom panels) or with scramble siRNA (si Scr, upper panels) and quantification (D) of the total force applied (nN) on fibronectin-coated polyacrylamide gel with a defined rigidity of 5 kPa. The additional silencing of β3 integrins in the β1−/−/icap-1−/− cells decimates the traction forces, confirming that, in absence of β1 integrin and ICAP-1, generation of strong cellular contractility at adhesion sites is dependent on β3 integrins. Scale bar, 10 μm. Error bars represent SD. N = 80 cells. ****, P value ≤ 0.0001. (E) FACS analysis of median fluorescence intensity of β3 integrin on cell surface in β1 integrin KO osteoblast cell lines. Error bars represent SD. (F) Western blot showing the efficiency of β3 integrin silencing in β1 integrin+/+-icap-1−/− and β1 integrin−/−-icap-1−/− cell lines. Source data are available for this figure: SourceData F2.

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