SFK activity is required for PP fragmentation in vivo. (A) DIC microscopy image sequences of PPs in vitro. Arrow, platelets released from PP stems. MKs were kept on a heated microincubator to keep the temperature at 37°C in serum-free medium and monitored using a DIC microscope system equipped with a ×40 oil objective lens with NA = 0.7. MKs treated with 10 µM S1P and dimethylsulfoxide (DMSO) (as vehicle for PP2) (upper row); MKs treated with 10 µM S1P and 10 µM PP2 (lower row). (B) S1P-induced PP fragmentation is dependent on SFK activity. The number of PPs with or without fragmentation observed by DIC microscopy in vitro over 1 hour in the indicated groups. Data are pooled from 5 to 7 independent experiments from each group (n = 25-54 per group). (C) Western blot analysis of Src and p-SFKs in WT MKs treated with vehicle (DMSO) or 10 µM dasatinib. Gapdh served as loading control. Representative of 3 independent experiments. (D) Percentage of PP fragmentation events observed by MP-IVM over 1 hour in the indicated groups. Data are pooled from 4 to 5 independent experiments from each group (n = 32-33 per group). (E) Role of SFKs in PP shedding in vivo visualized by MP-IVM. Dasatinib abolishes PP shedding in vivo (middle 2 and lower 2 rows). Images were captured through a ×20 water immersion objective lens with NA = 0.95 using a BioTech TriM Scope system. Arrowheads and arrows indicate the tips of PPs. Green, MKs and PP; red, sinusoids. The dashed line highlights the sinusoids. All scale bars represent 20 µm; time in minutes. All error bars represent SEM.