Figure 5. Dll-1 protein levels are negatively regulated by miR-1 in mES cells, and knockdown of Dll-1 expression promotes cardiac mesoderm and suppresses non-mesodermal gene expression.

(A) Dll-1 and Dll-4 mRNA levels, assessed by qRT-PCR, were somewhat higher in mES^miR-1 and mES^miR-133 cells than in controls.

(B) Immunostaining with Dll-4 or Dll-1 antibody showed equivalent Dll-4 protein levels in mES^miR-1, mES^miR-133 cells, and control mES cells; Dll-1 protein levels were lower in mES^miR-1 cells and higher in mES^miR-133 cells than wild–type mES cells.

(C) miR-1 expression caused a dose-dependent decrease in epitope (V5)-tagged Dll-1 protein levels by Western blot without affecting RNA expression of Dll-1 assessed by qRT-PCR (graph). Gapdh protein levels reflect equal loading of protein.

(D) Dll-1 mRNA levels, assessed by qRT-PCR, were 62% and 40% lower in response to two distinct short hairpin RNAs targeting Dll-1 mRNA (Dll-1^shRNA-1 and Dll-1^shRNA-2), compared to control cell line.

(E) EBs formed from Dll-1^control, Dll-1^shRNA-1 and Dll-1^shRNA-2 ES cells were scored for beating cardiomyocytes on days 8, 10, and 12 of differentiation. Beating cardiomyocytes appeared earlier and were more numerous in EBs from Dll-1^shRNA cell lines than in EBs from the control line.

(F) qRT-PCR analyses of Nkx2.5, Myogenin, Afp, and Nestin expression in EBs generated from Dll-1^control, Dll-1^shRNA-1 and Dll-1^shRNA-2 ES cells. Knocking down Dll-1 increased Myogenin expression, decreased Afp expression and sustained Nestin expression compared to controls.