Loss of B-Myb promotes differentiation and apoptosis.

<p>A) Relative mRNA levels were determined by qRT-PCR following knockdown of B-Myb. In these experiments, markers of differentiation (CoupTf, Fgf5, Sox1, Nestin, Sox17, Hdac2, Cdx2, Hand1) were either absent or at the limits of detection in undifferentiated cells, but following knockdown of B-Myb, several markers (CoupTf, Fgf5, Sox17, Cdx2 and Hand1) showed significant (p<0.05) changes in expression. B) Western analysis of Sox17, CoupTf and Hand1 confirmed that loss of B-Myb was associated with an up-regulation in markers of differentiation. C) Knockdown of B-Myb led to a significant reduction in transcripts encoding pluripotency markers Oct4 and Sox2 within 48 hours, but not in UTF1. A significant reduction in Nanog was generally only observed >72 hours after loss of B-Myb. All data were determined by qRT-PCR. D) Knockdown of B-Myb also led to a reduction in OCT4 and SOX2 proteins at 48 hours post-nucleofection that could be detected by western (n = 3). E) Over-expression of B-MYB, relative to the parental vector control, led to increased OCT4 and SOX2 protein abundance. F) Summary data for OCT4 and SOX2 expression in mES cells following over-expression of B-Myb (PGK-B-Myb). In these experiments, the over-expression of B-Myb was transient, as were the increases in Oct4 and Sox2. G) The WST-1 assay shows a significant segregation in the absorbance of the colormetric dye in D3 cells transfected with B-Myb shRNAs relative to controls. This segregation indicates a loss in cell viability in mES cells lacking B-Myb (p<0.05). H) Loss of B-Myb promotes apoptosis, but only 72 hours after nucleofection. No increased genomic DNA fragmentation (apoptosis) was ever observed at 24 or 48 hours following knockdown. Importantly, the DNA fragmentation could be inhibited by Z-VAD-FMK, a pan-caspase inhibitor, indicating that the observed DNA laddering was due to active caspases. *p<0.05 relative to NT controls (n = 3).