Fig. 5

HOXA9 represses the expression of HIF-1α and its downstream glycolytic genes by directly binding to the promoter regions. a, b The mRNA or protein expression of HOXA9, HIF-1α, HK2, GLUT1, and PDK1 was detected by qRT-PCR or western blot, respectively, in A431 cells after depletion of HOXA9 by two siRNAs or overexpression of HOXA9. The qRT-PCR data were normalized to GAPDH gene expression. In western blots, GAPDH was used as a loading control. c Known or predicted binding sites of HOXA9 (diamond) or HRE elements for HIF-1α (delta) in the promoter regions of HIF1A, HK2, GLUT1, and PDK1 as determined by rVista (https://rvista.dcode.org/). d Electrophoretic mobility shift assay (EMSA) was used to detect the direct association of purified HOXA9 protein with its binding sites on the promoter regions of HIF1A, HK2, GLUT1, and PDK1. The black arrows indicate the binding complex of the HOXA9 protein with the probe containing the known or predicted binding sites whereas the white arrows indicate the supershift generated by the association of the anti-HOXA9 antibody with the above complexes. Also, the specificity of the direct associations was supported by the loss of binding activities using mutated probes. e The binding enrichment of HOXA9 at the above binding sites on the promoter regions of HIF1A, HK2, GLUT1, and PDK1 was detected by ChIP-PCR after knockdown of HOXA9. f The binding enrichment of HIF-1α at the above binding sites on the promoter regions was detected after overexpression of HOXA9. Each experiment was performed in triplicate and data are presented as mean ± s.d. One-Way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001)