Figure 1.

FoxO3A contributes to the transcriptional regulation of the hypoxic response and is required for hypoxic repression of mitochondrial genes. (A) HeLa cell clones stably expressing FoxO3A shRNA or empty knockdown vector (Ctrl) were incubated for 16 h in normoxia or hypoxia (0.5% O₂) and analysed by immunoblotting. (B, C) RNA from three experiments performed with Ctrl and FoxO3A-KD#1 as in (A) was extracted, labelled, and hybridized to Affymetrix HG-U133 Plus 2.0 microarrays. Statistical analysis was applied to identify probe sets upregulated (B) or downregulated (C) by hypoxia in a FoxO3A-dependent manner. DAVID analysis for each group is shown below, respectively. (D) Overlay of Mitocarta gene list with the list of hypoxia-repressed FoxO3A-dependent genes. The list of genes in the overlap is shown below. (E) Validation of six FoxO3A-dependent hypoxia-repressed mitochondrial genes by qPCR. The experiment was performed as in (A). The results are normalized to 18S rRNA and are shown as fold repression in hypoxia. Error bars indicate mean±s.d. of three independent experiments, ^**P<0.01, ^***P<0.001 using Student's t-test.