Direct inhibition of p38MAPK signaling is sufficient to control primitive myeloid potential. (A) Western blotting analysis of total and phospho-p38 levels after treatment of day 5 EBs with DMSO (vehicle control) or SCIO469, a small-molecule inhibitor specific to the p38-α isoform. (B) Densitometric analysis from 3 separate experiments, with phospho-p38 normalized to total p38 protein expression levels. Primitive myeloid potential was analyzed using methylcellulose-based progenitor colony assays, which showed that (C) MacP and (D) MegaP potential after direct p38MAPK inhibition is enhanced to levels similar to those observed after BMP inhibition. Macrophage (E) and megakaryocyte (F) potential was analyzed in serum-free EB cultures after addition at day 4 of recombinant BMP4, with or without the p38-α inhibitor SCIO469. BMP4 treatment directly attenuated myeloid potential and this was rescued by simultaneous p38 inhibition. Furthermore, BMP4 increased p38 activation and decreased NOTCH pathway signaling, measured by western blotting analysis of phospho-p38 and cleaved NOTCH intracellular domain, respectively (G). Western blotting results are quantified from 3 separate experiments in panel H. Values are normalized to DMSO-treated (A-F) or untreated (G-H) controls; asterisks indicate significance, with *P < .05 and **P < .01, respectively. DMSO, dimethylsulfoxide.