Figure 2. RHOA^Y42C is a mutation with gain-of-function by stimulating stress fibers and focal adhesions.

A, Immunofluorescence analysis of NIH/3T3 fibroblasts stably expressing exogenous HA epitope-tagged RHOA WT and mutant proteins, stained with phalloidin to monitor stress fiber formation and anti-vinculin antibody to visualize focal adhesions (FA). Images are representative of three independent experiments. Scale bar = 10 μm. B, Quantitation of stress fiber formation in the cells from (A), by calculating the corrected total cell fluorescence (20 cells per condition, n = 3 independent experiments). To measure FA assembly in the cells from (A), the area of each FA (C), the number of FA per cell (D), and the eccentricity of each FA (E) was calculated based on the vinculin staining (1307 to 2295 FA in 14 to 20 cells per condition, n = 3). Data are mean ± S.E.M. F, CMFDA-labelled NIH/3T3 cells were allowed to adhere upon fibronectin for 1 h. Data are mean ± S.E.M. (n = 3). G, Images of cells during random migration were captured using a time lapse microscope at 1 frame/10 min for 16 h. Data are mean migration velocities ± S.E.M. (n = 4, 25 cells per experiment); ***P<0.001, **P<0.01, *P<0.05, ns, not significant; P values from one-way ANOVA with Tukey’s multiple comparison test. H, Representative immunofluorescence images for F-actin in organoids from mice with annotated genotypes. Phalloidin (in red) was used to visualize F-actin, DAPI (in blue) for the nucleus. Scale bar = 50 μm.