Figure 5.

ChIP assay for interactions of HIF-1α at its target genes in MRL/MpJ MDSPCs. Chromatin prepared from DMOG-treated MRL/MpJ MDSPCs was immunoprecipitated with control IgG and HIF-1α antibodies, followed by qPCR with gene-specific primers flanking HIF-1α responsive genomic sequence using ChIP DNA. A–C) Interactions of HIF-1α are indicated by % bound input: 3 regions in Pax7, the promoter region (Prom.), first intron, and 3′UTR as control region (A); the Vegfa promoter (B); and the Glut1 promoter that harbors HIF-1α –responsive genomic sequence and is indicated by a solid triangle (C). D) ChIP assay for HIF-1α occupancy at the Pax gene in CoCl₂-treated MRL/MpJ MDPSCs. E) Schematic of a construct containing the 1.2-kb mouse Pax7 promoter driving the expression of Zsgreen (GFP). F, G) Fluorescent microscopic images taken after transient transfection with the Pax7-Zsgreen reporter gene in the myoblast cell line C2C12 (F) and HEK 293 cells (G), each in the presence of DMOG (1 mM) or CoCl₂ (200 μM), and coexpression of mutant HIF-1α after transfection as indicated. Scale bars, 10 μm. H) Level of HIF-1α in MRL/MpJ MDSPCs after transducing with lenti-nshRNA and lenti-shRNA followed by precondition to hypoxic conditions for 24 h was analyzed with Western blot using the cell lysates. I) Quantitative gene expression showing mRNA levels of myogenic stem cell markers Pax7, Hes1, and Notch3 in nshRNA and HIF-1α shRNA MDSPCs from MRL/MpJ mice.