Cholesterol metabolic process was the major pathway regulated by hypoxia-induced TET1 and activation of INSIG1 expression by hypoxia, which was abolished by TET1 knockdown using real-time PCR analysis. (a) Scatter plot comparing transcriptome profile of hypoxia sample with scrambled knockdown vs. hypoxia sample with TET1 knockdown in FADU cell lines. Gene expression was measured by FPKM. The genes with the fold change >2 or <0.5 were denoted as black dots. The 1,044 genes regulated by TET1 under hypoxia were highlighted by red dots. The square dot indicating the INSIG1 gene was pointed out. (b) Venn diagram showed the overlapping group of genes (98) that had both differential RNA expression and differential 5hmC-enriched promoter regions. (c) Gene Ontology analysis of the genes with differential 5hmC-enriched regions and differential RNA expression. The top 10 enriched biological processes based on their P values were shown. Cholesterol metabolic process ranked the most significant group of genes regulated according to RNA expression and 5hmC peak levels. (d) Activation of INSIG1 expression by hypoxia in H1299 cells using real-time PCR analysis. N: normoxia; H: hypoxia. (e) Knockdown of TET1 abolished the activation of INSIG1 expression induced by hypoxia in H1299 cells using real-time PCR analysis. The asterisk (*) indicates statistical significance (P <0.05) between experimental and control clones. The H1299 vector or H1299 scrambled control clone under normoxia was chosen as the control condition in (d) and (e). Error bars indicate standard deviations (s.d.) of duplicate mRNA levels by real-time PCR analysis (d,e).