The constitutively active DIAPH1 (mDia1ΔN3) rescues the effects of shRNA-induced knockdown of DIAPH1. (A) CD41⁺GFP⁺Cherry⁺ MK populations double-infected with 2 lentiviral vectors containing GFP or Cherry were purified by flow cytometry. In each combination (top left: SCR plus control; bottom left: sh8+control; top right: SCR+mDia1ΔN3; bottom right: sh8+mDia1ΔN3), the CD41⁺ population was first gated and then the double-labeled population was obtained by gating the GFP⁺ Cherry⁺ population in the previously gated CD41⁺ population. (B) Quantitative RT-PCR showing the levels of endogenous and exogenous DIAPH1 mRNA in the double-infected populations purified by flow cytometry (3 independent experiments). (C) PPF was counted in the double-transduced MK population CD41⁺GFP⁺Cherry⁺. The increase of PPF caused by the shRNA targeting DIAPH1 was abolished by mDia1ΔN3 expression (4 independent experiments; PPF in SCR plus control MKs was used as a normalized baseline). (D) Tubulin polymerization assays in CD41⁺ MKs show that Rock or myosin II inhibition increased tubulin polymerization, but disturbed stress fiber formation. Top: Control; middle: Blebbistatin; bottom: Y27632. MKs were stained for tubulin (α and β, green) and phalloidin-TRITC (red); DNA was stained with TOTO (blue). (E) Blebbistatin or Y27632 incubation increased α-tubulin or β-tubulin fluorescence area relative to total cell area (%Area). There were 30 to 40 cells in each condition were quantified in 3 independent experiments using Image J. The data are presented as mean ± standard error of the mean: control (5.77 ± 0.85); blebbistatin (13.63 ± 1.99); Y27632 (13.07 ± 1.75). *P < .02; **P < .005.