SMAD-independent BMP control of myeloid potential is dependent on NOTCH, associated with increased expression of Δ-like ligands. (A) Quantitative RT-PCR analysis of Δ-like genes Dll1 and Dll3 in day 5 EBs after 24-hour treatment with dox to deplete Smad5 and Smad1 (black bar); LDN only to inhibit BMP signaling (gray bar); or dox + LDN demonstrating that enhanced expression is SMAD-independent (striped bar). (B) qPCR analysis of NOTCH effector genes 24 hours after BMP inhibition with LDN. (C) Western blotting analysis of changes in NOTCH activation via cleaved receptor intracellular domain levels (NICD), 24 hours after BMP inhibition by LDN50 in day 4 EBs. (D) Western blotting analysis of NICD 24 hours after 5 μM DAPT treatment of day 4 EBs. Representative blots are shown with densitometric analysis of relative levels compared with untreated controls. Methylcellulose colony assay analyses of (E) macrophage and (F) megakaryocyte potential under conditions of LDN50 treatment alone (compare small striped gray bars to black control bars) or in combination with 5 μM DAPT (compare gray bars to large striped bars), with each experiment performed at least 3 separate times. (G) qPCR analysis of transcript levels for myeloid regulatory transcription factor C/EBPα on day 8 of EB differentiation after day 4 treatment with LDN alone (compare black control bar to gray bar), or cotreatment with LDN + 5 μM DAPT (compare gray bar to striped bar). Values in qPCR assays are reported as mean ± SEM from at least 3 experimental repeats, normalized to untreated controls, and compared with Gapdh as a reference gene. In all experiments, *P < .05 and **P < .01.