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. 2016 Oct 24;5(10):e263. doi: 10.1038/oncsis.2016.63

Figure 3.

Figure 3

GPR133 expression is upregulated by hypoxia. (a) Hypoxia upregulates GPR133 mRNA in 6/8 primary cultures (n=3 measurements per culture; ANOVA F(1,12)=14.82; P<0.003). (b). Analysis of the GPR133 genomic locus reveals numerous HRE motifs, including immediately upstream of the TSS. The schematic also shows primers used for Hif1α ChIP-PCR experiment (green arrows). (c) Western blot shows effects of Hif1α knockdown (HIF1A-KD) on Hif1α protein levels in two primary cultures. β-Actin was used as a loading control. (d) Hif1α knockdown downregulates HIF1A and GPR133 mRNA under normoxic (i, HIF1A: n=3 experiments per culture, t-test, P<0.002; and ii, GPR133: n=3 experiments per culture t-test, P<0.05) and hypoxic conditions (iii, HIF1A: n=3 experiments per culture, t-test, P<0.02; and iv, GPR133: n=3 experiments per culture, t-test, P<0.04) in two primary cultures: GBML8 (top row) and GBML20 (bottom row). (e) Fold enrichment (i) and percent of input (ii) representations of ChIP-PCR using Hifα antibody reveal that GPR133′s promoter region containing the HRE binds Hifα directly (i, n=3 primary cultures, two-tailed t-test, P<0.001; ii, n=3 primary cultures, one-tailed t-test, P<0.05). CA9 promoter was used as a positive control (i, n=3 primary cultures, two-tailed t-test, P<0.008; ii, n=3 primary cultures, one-tailed t-test, P<0.04). Immunoglobulin G (IgG) alone was used as a negative control.