Stiff matrix promotes higher intracellular signaling. (A) (Left) Higher FAK phosphorylation upon MK adhesion on stiff substrates. Red, F-actin; green, P-FAK. Scale bars, 10 μm. (Right) Quantification of P-FAK positive puncta per MK; mean ± SEM (20-25 MKs, 2 independent experiments). (B) (Left), RhoA–guanosine triphosphate immunolabeling. Scale bars, 15 μm. (Right) Higher RhoA activity upon MK adhesion on stiff substrate; mean ± SEM (53 MKs for 1.5 kPa, 69 MKs for 90 kPa, and 27 MKs for glass surface; 3 independent experiments). Ordinary 1-way ANOVA with Tukey multiple comparisons test.