Figure 6. Delta-1 up-regulates expression of Wnt receptors via Notch signaling.

A. Enriched HPCs were cultured in wells of a 24-well plate coated with Delta-IgG or control IgG with or without GSI (5nM) for 24 h. Expression of fzd receptors and Wnt-targeted genes were evaluated in quadruplicates by qRT-PCR. Hes1 was included as a positive control. B. 32D cells were transfected with a control non-target pool of siRNAs or CBF-1 siRNA. Inhibition of CBF1 expression was confirmed by Western-blotting 48 hr after transfection (inset). Expression of fzd and Wnt-targeted genes was measured in quadruplicates by qRT-PCR. C. qRT-PCR. was used to determine the expression of fzd and Wnt-targeted genes in wild type or Notch-1-deficient ES cells. In panels A–C mean ± SE from 3 experiments are shown. D. ChIP assay was performed in 32D cells using antibodies against CBF-1 or control IgG as described in Experimental Procedures. Recruitment of potential CBF-1 binding sites within the fzd10 promoter by CBF-1 protein was evaluated by qPCR and presented as fold increase over input DNA. Two experiments with the same results were performed. E. HPCs were transduced with GFP or Notch-IC-GFP vectors and cultured in complete medium containing 20 ng/ml GM-CSF for 24 h. GPF-positive cells were sorted and the expression of fzd and Wnt-targeted genes was evaluated in quadruplicates by qRT-PCR. Two experiments with the same results were performed. F. HPCs were cultured in wells of a 24-well plate coated with 7.5 ng/ml control IgG or Delta-1 with or without Wnt inhibitor WIF-1 (750 ng/ml) for 24h. Expression of Wnt-targeted genes was measured in quadruplicates by qRT-PCR. Fold increase over background (gene expression in HPC cultured with control IgG) is shown.