RhoA is overactivated in FLNa-deficient MKs. (A) Immunofluorescence staining of G-actin (red) and F-actin (green) after adhesion on fibrinogen. The nucleus is stained with 4′,6-diamidino-2-phenylindole (blue); scale bar = 30 μm. (B) Quantification of the percentage of stress fiber–forming MKs for each edited clone. Results are presented as mean ± standard error of the mean; each point represents 1 independent experiment. Data were analyzed by performing 1-way analysis of variance (ANOVA) followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). (C) FRET analysis for RhoA activation on different substrates: representative image, scale bar = 10 μm (i) and adhesion on fibrinogen (n = 4), collagen 1 (n = 3), fibronectin (n = 6), VWF (n = 3), and vitronectin (n = 5) (ii). (D) FRET analysis of RhoA activation on fibrinogen of all edited clones: WT and mutant clones (n = 8), del1 and del4 clones (n = 6), and del2 and del3 clones (n = 5). (C-D) At least 15 cells per condition were analyzed. Data were analyzed by performing ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method); all experiments were performed at day 15 or 16 of MK differentiation. *P < .05, **P < .01, ***P < .001.