Figure 5.
Integrin β3 is required for FN stiffness sensing and fibrillogenesis. (A) Immunolabeling of mouse BM sections showing co-localization between FN (green) and Itgb3 (left) or Itgb1-positive MK (right) (red labeling). Representative of at least 3 independent sections. Bar is 10 μm. (B) Number of WT, Itgb1–/–, Itgb3–/–, and Itgb1b3–/– MK adhered for 2 hours onto PDMS substrates of various stiffness coated with FN 50 μg/mL using microcontact printing (circular patterns of 200 μm diameter); 3 independent experiments performed simultaneously with the 4 genotypes, each dot represents the mean of 5 replicates; ordinary 1-way ANOVA with Tukey multiple comparisons test. (C) Measurement of the visible surface of MK adhered for 5 hours, observed by bright field microscopy. Ordinary 1-way ANOVA with Tukey multiple comparisons test; §P < .001 comparing the control with the substrate of same stiffness. (D) (i) F-actin labeling of WT and Itgb3–/– MK adhered on FN-coated glass coverslip for 5 hours. Scale bar, 10 μm; (ii) positive F-actin spreading area per MK; (iii) circularity; (E) (Left) Confocal images showing immunofluorescence labeling (arbitrary units) of RhoA-GTP (yellow) and P-FAK (green) in control, Itgb1–/– and Itgb3–/– MKs following 5 hours adhesion onto soft and stiff FN substrate; (middle) quantification in arbitrary units of the RhoA-GTP labeling intensity; n = 30 to 46 MKs from 3 independent experiments; (right) quantification of the number of P-FAK positive puncta per MK; n = 28 to 31 MKs from 3 independent experiments. Ordinary 1-way ANOVA with Šidák multiple comparisons test. (F) (Left) Representative 2 confocal images showing biotin-FN (revealed by FITC-streptavidin, green) at the periphery of the control, Itgb1–/–, Itgb3–/–, and Itgb1b3–/– MKs adhered for 5 hours on 90 kPa substrate. Scale bar, 10 μm. (Right) Quantification of the proportion of MK presenting FN accumulation at anchorage points, peripheral accumulation and visible individual fibrils (n = 3 independent experiments; 60-63 MKs per genotype).

Integrin β3 is required for FN stiffness sensing and fibrillogenesis. (A) Immunolabeling of mouse BM sections showing co-localization between FN (green) and Itgb3 (left) or Itgb1-positive MK (right) (red labeling). Representative of at least 3 independent sections. Bar is 10 μm. (B) Number of WT, Itgb1–/–, Itgb3–/–, and Itgb1b3–/– MK adhered for 2 hours onto PDMS substrates of various stiffness coated with FN 50 μg/mL using microcontact printing (circular patterns of 200 μm diameter); 3 independent experiments performed simultaneously with the 4 genotypes, each dot represents the mean of 5 replicates; ordinary 1-way ANOVA with Tukey multiple comparisons test. (C) Measurement of the visible surface of MK adhered for 5 hours, observed by bright field microscopy. Ordinary 1-way ANOVA with Tukey multiple comparisons test; §P < .001 comparing the control with the substrate of same stiffness. (D) (i) F-actin labeling of WT and Itgb3–/– MK adhered on FN-coated glass coverslip for 5 hours. Scale bar, 10 μm; (ii) positive F-actin spreading area per MK; (iii) circularity; (E) (Left) Confocal images showing immunofluorescence labeling (arbitrary units) of RhoA-GTP (yellow) and P-FAK (green) in control, Itgb1–/– and Itgb3–/– MKs following 5 hours adhesion onto soft and stiff FN substrate; (middle) quantification in arbitrary units of the RhoA-GTP labeling intensity; n = 30 to 46 MKs from 3 independent experiments; (right) quantification of the number of P-FAK positive puncta per MK; n = 28 to 31 MKs from 3 independent experiments. Ordinary 1-way ANOVA with Šidák multiple comparisons test. (F) (Left) Representative 2 confocal images showing biotin-FN (revealed by FITC-streptavidin, green) at the periphery of the control, Itgb1–/–, Itgb3–/–, and Itgb1b3–/– MKs adhered for 5 hours on 90 kPa substrate. Scale bar, 10 μm. (Right) Quantification of the proportion of MK presenting FN accumulation at anchorage points, peripheral accumulation and visible individual fibrils (n = 3 independent experiments; 60-63 MKs per genotype).

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