Expression of a constitutively active DIAPH1 (mDia1ΔN3) inhibits PPF. (A) CD41⁺GFP⁺ MKs were sorted by flow cytometry after infection with mDia1ΔN3 (right) or with the empty lentiviral vector (left) (pRRL-EF1α-PGKGFP, control) at day 7 of MK culture. (B) Quantitative RT-PCR for endogenous and exogenous mRNA expression in sorted CD41⁺GFP⁺ MKs. Total DIAPH1 mRNA expression was threefold to fourfold higher in MKs transduced with mDia1ΔN3 relative to controls (cells transduced with pRRL-EF1α-PGKGFP) in 3 independent experiments. (C) PPF measured from day 12 to day 14 in MK culture was lower in MKs expressing mDia1ΔN3 than in controls (cells transduced with pRRL-EF1α-PGKGFP) in 4 independent experiments. (D) mDia1ΔN3-expressing MKs (bottom) often presented proplatelets with shorter extensions and less branching than controls (top); phase contrast images with a ×20 lens. (E) Proplatelet area was measured by Image J in 3 independent experiments. The data are presented as mean ± standard error of the mean. The mDia1ΔN3-infected MKs (0.0018 ± 0.0002 mm²; n = 32) had a lower proplatelet area than controls (0.0008 ± 0.0001 mm²; n = 30). (F) Proplatelet branch numbers were counted in 3 independent experiments. The mDia1ΔN3-infected MK (6.57 ± 0.94; n = 32) showed less branching than the controls (cells transduced with pRRL-EF1α-PGKGFP) (2.81 ± 0.42; n = 35). *P < .01; **P < .0008.