Differential roles of WAVE1 and WAVE2 in integrin-mediated lamellipodia formation in ES cell–derived MKs. (A) MK progenitors were infected with lentivirus containing shRNA on day 5 for 14 hours. Sorted α_IIb⁺ c-Kit⁺ cells were further cultured with OP9 stromal cells. Progenitors expressing GFP on day 9 were sorted and lysed. The lysate was subjected to SDS–gel electrophoresis on Western blotting. On day 12, MKs were plated on fibrinogen for 60 minutes (i, iii) or 120 minutes (ii, iv) in the presence of 100 nM PMA, and fixed and stained with rhodamine-conjugated phalloidin to mark F-actin. (B) GFP⁺ or GFP⁻ cells transduced with control shRNA, WAVE1 shRNA, or WAVE2 shRNA were assessed by the significance of morphology. A total of 4 independent experiments were performed. (C) MKs on fibrinogen were fixed after washing with PBS once, twice, or 3 times and were stained with α_IIb. Increased numbers of washing procedure promoted dissociation of MKs. Data are means (± SD; n = 3). (D) Control MKs or WAVE2^−/− MKs transduced with shRNA of scrambled sequence and GFP, and 2 representative fibrinogen-adherent WAVE2^−/− MKs transduced with shRNA of WAVE1 and GFP (i,ii) are shown.