Effects of the double inhibition of DIAPH1 and Rock/myosin II on PPF. (A) Left: Western blot performed on CD41⁺ MKs at day 8 of culture revealed Glu-tubulin, Tyr-tubulin, and acetylated-tubulin expression after ROCK (Y27632, 10 μM) or myosin II inhibition (blebbistatin 25 μM) with overnight incubation. Right: The ratio of Glu-tubulin to Tyr-tubulin and quantification of acetylated-tubulin expression relative to HSC70 showed a clear increase of stable Glu-tubulin, but only a slight increase of acetylated-tubulin. (B) The sh7-, sh8- or SCR-transduced MKs were incubated overnight just before PPF (at day 10 of culture) with inhibitors of Rock (Y27632, 10 μM) or myosin II (blebbistatin 25 μM). Inhibition of Rock and of myosin II combined with DIAPH1 knockdown by shRNA increased PPF even more in 3 independent experiments. (C) Knockdown of both MYH9 and DIAPH1 by shRNA increased PPF more the knockdown of either alone in 3 independent experiments. MKs were double-infected with 2 lentiviral vectors containing GFP (shMYH9 or SCR control) or Cherry (sh8 or SCR control). CD41⁺GFP⁺Cherry⁺ populations (SCR plus SCR, SCR plus sh8, SCR plus shMYH9, or sh8 plus shMYH9) were purified by flow cytometry. (D) Quantitative RT-PCR showing DIAPH1 and MYH9 mRNA levels in double-infected MK populations (SCR plus SCR, SCR plus sh8, SCR plus shMYH9, or sh8 plus shMYH9). Cells were isolated by flow cytometry in 3 independent experiments. (E) Top: Western blots of sh7-, sh8-, or SCR-transduced MKs incubated overnight with inhibitors of ROCK (Y27632, 10 μM) or myosin II (blebbistatin, 25 μM) at day 8 of culture. Glu-tubulin levels increased even more after DIAPH1 knockdown combined with Rock/myosin inhibition. A 10% SDS-PAGE gel was used for protein separation. Bottom: The ratios of DIAPH1/HSC70 and Glu-tubulin/Tyr-tubulin showed a clear decrease of DIAPH1 expression and an increase of stable Glu-tubulin after DIAPH1 knockdown combined with inhibitor. DMSO, dimethylsulfoxide. *P < .05; **P < .005.