Soft matrix is preferred for proplatelet formation. (A) (Left) Bright field image of MK forming proplatelets after 24 hours adhesion on the 1.5 kPa FN micropattern. Scale bar, 50 μm. (Right) Quantification of MK forming proplatelets depending on the matrix stiffness. Ordinary 1-way ANOVA with Tukey multiple comparisons test, mean ± SEM, n = 3 independent experiments, each dot is the mean of 9 replicates. (B) Decreased spreading after 24 hours adhesion. (i) xy and xz view of a MKs adhered for 5 or 24 hours; red, F-actin, blue, nucleus. Scale bar, 10 μm; (ii) time-lapse of a MK expressing Lifeact-GFP (green); acquisition was one every hour and started 6 hours after seeding; bar is 10 μm. (iii) Co-relation between spreading area and height of MKs adhered for 5 hours or 24 hours on FN-coated glass surface. Mean ± SEM from 25 MKs in 2 independent experiments. (C) Impact of blebbistatin on proplatelet formation on the different substrate stiffness after 24 hours adhesion; n = 3 independent experiments; each dot is the mean of 9 replicates ± SEM. For the vehicle group, statistical analysis was performed with ordinary 1-way ANOVA and Tukey multiple comparisons; ∗∗∗∗P < .0001. Comparison between vehicle and blebistatin was with 2-way ANOVA and Šidák multiple comparisons; ^§P < .05, ^§§P < .01, comparison between vehicle and blebbistatin treatment for a same stiffness.