Figure 3.

IH leads to HIF-1α-dependent increase in NOX4 mRNA expression in PC12 cells. A: Nox4 mRNA expression in lysates from PC12 cells treated with lentiviral HIF-1α shRNA or scr shRNA and exposed to 60 cycles of IH (IH₆₀) or normoxia (N) (means ± SE; n = 4 independent experiments with duplicates). B: two putative HRE HIF-binding sites (red, boxed), one (HRE1) found at base pairs −147 to −151 relative to the transcription start site (shown in blue), and a second (HRE2) found approximately +167 bases downstream from the transcription start site of the Nox4 gene. C: ChIP assay of PC12 cells exposed to IH₆₀ or normoxia (N) using antibodies against HIF-1α. Enrichment of each sequence in the immunoprecipitates relative to the starting lysate was determined by qRT-PCR. Data are presented as means ± SE from two independent experiments with triplicates. **P ≤ 0.05; n.s. = not significant (P > 0.05) as determined by Mann-Whitney test. ChIP, chromatin immunoprecipitation; HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; IH, intermittent hypoxia; KDM, lysine demethylases; Nox4, NADPH oxidases; PC, pheochromocytoma; qRT-PCR, quantitative reverse transcription-PCR; scr, scramble.