Control of myeloid potential by BMP signaling is SMAD-independent. (A) Titration of LDN compound and western blotting analysis of SMAD1/5/9 phosphorylation compared with total SMAD1 expression shows dose dependence of BMP signaling in day 4/5 EBs, with ∼50% inhibition achieved under conditions of 0.01 μM LDN (B, henceforth noted as LDN50). Analysis of myeloid potential by methylcellulose colony assays to examine (C) macrophage, (D) megakaryocyte, and (E) mixed lineage progenitors shows persistence of myeloid phenotype after LDN treatment regardless of whether baseline is untreated control (black bar compared with thin-striped bar) or dox-induced Smad5/Smad1 knockdown (gray bar compared with wide-striped bar). (F) Effects of BMP signaling inhibition on myeloid potential were examined on progenitors by sorting Flk1⁺ cells from day 3 EBs, and reculturing them 24 hours later in the presence of LDN50. Shown is a representative sort. (G) Macrophage potential was measured; gray bars correspond to FLK1⁻ cells, striped bars to a 50/50 mix of FLK1⁻ and FLK1⁺, and black bars to cultures containing FLK1⁺ only. Colony assays were each performed at least 3 separate times, with samples counted in triplicate. *P < .05; **P < .01.