Expression of a constitutively active DIAPH1 (mDia1ΔN3) increases stress fiber formation and decreases tubulin polymerization and stability. (A) Left: A stress fiber formation assay was performed in sorted CD41⁺GFP⁺ MKs transduced with mDia1ΔN3 or with an empty vector as described in “Methods.” Right: A higher percentage (50.7%) of mDia1ΔN3 MKs showed stress fiber formation than did controls (38.5%) in 3 independent experiments. *P < .01. (B) Left: CD41⁺ MKs infected by mDia1ΔN3 or the control vector were plated on collagen-coated slides and treated for 1 hour with nocodazole. Tubulin polymerization assays were performed 10 minutes after recovery. The mDia1ΔN3-infected MKs showed lower tubulin polymerization relative to controls. Right: MKs infected by mDia1ΔN3 showed lower α-tubulin (top) or β-tubulin fluorescence area (bottom) relative to total cell area (%Area). We quantified 37 cells in each condition in 3 independent experiments using Image J. The data are presented as mean ± standard error of the mean. α-Tubulin: control: 10.10 ± 1.14; mDia1ΔN3: 6.64 ± 0.73. β-Tubulin: control: 6.14 ± 0.69; mDia1ΔN3: 3.82 ± 0.48. *P < .01; *P < .007. (C) Western blot analysis and quantification were performed in sorted CD41⁺GFP⁺ MKs transduced with mDia1ΔN3 or with an empty vector in 3 independent experiments showing that mDia1ΔN3 expression decreased the ratio between Glu-tubulin and Tyr-tubulin, but did not change the ratio between PMLC2 and MLC2. A 10% SDS-PAGE gel was used for protein separation. *P < .03.