Effects of collagen nano-mechanical properties on MK behavior. (Ai-ii) Phase-contrast images of MKs seeded for 16 hours in adhesion on collagens. Scale bar represents 100 μm. (iii-iv) Stress fiber formation was analyzed after actin (tetramethylrhodamine isothiocyanate [TRITC]-phalloidin, red) staining in immunofluorescence, whereas proplatelet formation was evaluated using an anti–α-tubulin (green) antibody. Nuclei were counterstained with Hoechst 33288 (blue). Images were acquired using a 63×/1.25 UPlanF1 oil-immersion objective. Scale bar represents 20 μm. (v-vi) Scanning electron micrographs of spread MKs on type I collagen and a platelet-releasing MK on N-acetylated type I collagen. Images were acquired with a Cambridge Stereoscan 440 microscope (Leica Microsystems) at 17.5 kV and a magnification of 2000×. Scale bar represents 4 μm. (B) Evaluation of cell spreading after 2 and 16 hours of adhesion. Cells were fixed and stained with TRITC-phallodin. Cells exhibiting stress fibers were counted and presented as mean ± SD of 3 different experiments. P < .05. SHG indicates second harmonic generation. (C) Proplatelet formation was analyzed after 16-hour adhesion on different collagens or in MKs maintained in suspension (none). P < .05. (D) Migration of MKs in transwell plate after 16 hours of incubation; 8-μm polycarbonate pore filters were coated with native and N-acetylated collagen or with BSA (none), and cells were counted after CD41 staining in immunofluorescence. Results are reported as percentage of migrated cells relative to control of 3 different experiments ± SD. (Ei-ii) Assembly of FN under static conditions. MKs were incubated with 25 μg/mL of FITC-labeled FN and then seeded on different matrices. FN fibrillogenesis was then visualized in immunofluorescence using a 63×/1.25 UPlanF1 oil-immersion objective. Hoechst 33288 was used for nuclei staining (blue). Scale bar represents 10 μm. (eiii-iv) Alternatively, cells were removed using deoxycholic acid (DOC) and the underlying matrix stained with an anti-FN antibody (red) and visualized in immunofluorescence, using a 40×/0.75 oil-immersion objective. Scale bar represents 20 μm. (F) Relocation of endogenous FN in MKs after 2- (left panels) and 16-hour (right panels) adhesion on collagens. Cells were stained for FN (red), α-tubulin (green), and Hoechst for nuclei (blue) using a 63×/1.25 UPlan oil-immersion objective. Scale bar represents 10 μm. (G) Distribution of Young modulus values of MKs in adhesion on different collagen samples. Three different experiments were performed, and at least 5 cells for sample were analyzed. P < .05. (H) Analysis of α2β1 and GPVI-dependent pathways in adhering MKs. Rho guanosine triphosphate pull-down experiments and immunoblot analysis of endogenous MLC2, Syk, and Src phosphorylation levels in MKs adhering to native and N-acetylated type I collagen after 16-hour incubation, representative of 3 different experiments. Actin, tubulin, and nonmuscle myosin IIA were revealed to demonstrate equal protein loading.xxxxx.