DIAPH1 knockdown decreases stress fiber formation, but increases tubulin polymerization and stability. (A) MKs transduced with different shRNAs (SCR, sh7, or sh8) after 1 hour of adhesion onto a fibrillar collagen I substrate for a stress fiber formation assay. MKs infected with sh7 (middle) and sh8 (right) showed fewer stress fibers than did control (SCR) (left). (B) MKs transduced with sh7 and sh8 showed 16.3% and 16.1% stress fiber formation, respectively, relative to 26.3% for the control (SCR) in 3 independent experiments. (C) DIAPH1 knockdown did not modify the ratio of pMLC2/MLC2 or of acetylated-tubulin/total tubulin. A 10% SDS-PAGE gels was used for protein separation (left). Three independent experiments were quantified with Image J (right). (D) CD41⁺ MKs infected with SCR, sh7, or sh8 were plated on collagen-coated slides for 1 hour and then treated for another hour with nocodazole. Tubulin polymerization assays were performed 10 minutes after washing. MKs infected with sh7 (middle) or sh8 (bottom) show increased tubulin polymerization relative to the control (top). The α-Tubulin is labeled in blue and β-tubulin in red. (E) MKs infected with sh7 or sh8 showed a greater α-tubulin or β-tubulin fluorescence area relative to total cell area (%Area). We quantified 30 to 40 cells for each condition in 3 independent experiments using Image J. The data are presented as means ± standard error of the mean. Top: α-tubulin (SCR: 8.04 ± 1.02; sh7: 12.96 ± 1.83; sh8: 12.37 ± 1.69). Bottom: β-Tubulin (SCR: 6.60 ± 0.96; sh7: 14.32 ± 1.61; sh8: 12.15 ± 1.58). (F) Western blots revealed that MKs infected with sh7 or sh8 presented a higher proportion of Glu-tubulin and a lower proportion of Tyr-tubulin relative to SCR controls. A 10% SDS-PAGE gel was used for protein separation (top). The expression ratios between DIAPH1 and HSC70 and between Glu-tubulin and Tyr-tubulin were quantified in 3 independent experiments using Image J (bottom). 3D, 3-dimensional. *P < .05; **P < .005.