DIAPH1 knockdown by shRNA increases MK PPF. (A) Western blots revealed that shRNA (sh7 and sh8) targeting of DIAPH1 decreased DIAPH1 protein level (left), but not DIAPH2 (middle) and DIAPH3 protein (right) expression, relative to control shRNA (SCR). A 10% SDS-PAGE gel was used for protein separation. (B) DIAPH1 (left), DIAPH2 (middle), or DIAPH3 (right) protein levels (relative to HSC70) were analyzed by Image J in 3 independent experiments. Only DIAPH1 expression was decreased by sh7 and sh8. (C) RT-PCR (normalized to HPRT) results showed that only the DIAPH1 mRNA level was decreased by sh7 and sh8 (4 independent experiments). (D) DIAPH1 knockdown resulted in a marked increase in MK PPF relative to the control (scr) (5 independent experiments). (E) Phase-contrast microscopy images (lens ×20) of day 13 culture of control (SCR), sh7-, or sh8-infected MKs. There was more PPF in sh7- or sh8-infected cells. NS, no significant difference. *P < .05; **P < .005.