PRMT1-mediated suppression of megakaryocytic differentiation is dependent on activation of the p38 MAPK. K562 cells were transiently transfected with FLAG-p38α and empty vectors and analyzed for megakaryocytic differentiation 96 h after PMA treatment ( A ). Stable cell clones (p38α KD) transfected simultaneously with two p38α shRNAs ( sh-1 and sh-2 ) were selected, and the protein levels were examined by Western blot analysis ( B, upper panel ). Luc , luciferase shRNA; GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Reduced expression of p38α enhanced megakaryocytic differentiation ( B, lower panel ). Treatment with the p38 inhibitor SB203580 ( SB ) greatly enhanced megakaryocytic differentiation in both parental K562 and HA-PRMT1-expressing R2-1 and R2-3 cells when analyzed 96 h after PMA treatment ( C ). The specific inhibitor of p38 MAPK, SB203580, was added 30 min before stimulation with PMA. Adherent cells were examined by phase contrast microscopy 96 h after PMA treatment ( D, left panel ) and quantified ( D, right panel ). Transient knockdown of p38α with either p38α sh-1 or p38α sh-2 shRNAs enhanced megakaryocytic differentiation in both K562 and R2-1 cells ( E ). Ectopic expression of p38α by transient transfection suppressed megakaryocytic differentiation in both K562 and PRMT1-knockdown cells (PRMT1 KD-1 and KD-2) ( F ). Cytological changes were detected by modified Wright-Giemsa staining ( F, upper panel ) and quantified ( F, lower panel ). HA-PRMT1 was transiently expressed in stable p38α-knockdown clones (p38α KD-1 and p38α KD-2) ( G ). Ectopic expression of HA-PRMT1 could no longer suppress megakaryocytic differentiation in p38α-deficient cells. Cells stably transfected with the luciferase shRNA ( Luc ) were used as a negative control. All experiments were performed at least three times, and data are presented as means ± S.E.; *, p