IRSp53 may be associated with Rac-dependent lamellipodia formation in adherent MKs but not with Rac-independent MK maturation. (A) ES cell–derived progenitors expressing GFP via control or IRSp53 knockdown were sorted. The lysate was subjected to SDS–gel electrophoresis on Western blotting. On day 12 of the differentiation protocol, ES cell–derived MKs transduced with shRNA of IRSp53 and GFP were plated onto 100 μg/mL fibrinogen for 60 minutes. IRSp53 knockdown cells exhibited filopodia-dominant features in the absence or presence of 100 nM PMA (arrowheads), while control megakaryocytes displayed peripheral lamellipodial ruffles. Scale bar equals 20 μm. Summarized results are the same as those in Figure 6E. Data are means (± SD). (B,C) Cells were prepared from E13.5 FL as described in “Cell culture.” Cells were exposed for 14 hours to a lentivirus encoding both several shRNAs and GFP on day 0. When studied on day 4, approximately 40% of the cells exposed to virus were successfully transduced, as assessed by GFP expression (not shown). The transduction of WAVE2 or Abi1 shRNA reduced (B) MK size and (C) proplatelet numbers throughout observation until day 7 of culture. Insets in panel B depict the morphology of proplatelets expressing GFP. This experiment is representative of 3 performed. Magnification, ×40. Numbers of proplatelets from FL-derived primary MKs on day 4 and day 7 (C). Data are means (± SD). *P < .05 versus control for day 4 or day 7. See “Image acquisition for platelet and MK spreading” for complete image acquisition information.