Figure 2. Microbial metabolites decrease HIF-2α expression and activity.

A) Schematic of duodenal metabolite extraction from wild type SPF mice and HIF-2α expression assay in intestinal cell lines, and B) HIF-2α Western analysis of DFO or 1% O₂ pretreated (4 hours) HCT116 cells followed by treatment with duodenal extracts (Ext) for 16 hours. C) Schematic of aqueous and organic extraction of duodenal and fecal metabolites from wild type SPF mice and HIF-2α expression assay in intestinal cell lines. HIF-2α Western analysis (D and E) in DFO- or 1% O₂ treated HCT116 cells or co-treated with organic extracts (Ext) for 16 hours. (F) HIF-2α Western analysis in DFO-treated HCT116 cells co-treated with Ext, heated (95°C) Ext [Ext(H)] (upper panel) or trypsin-digested Ext [Ext(T)] (lower panel) from fecal organic extracts. G) HIF-2α Western analysis in DFO-treated HCT116 cells or co-treated with organic (upper panel) and aqueous (lower panel) extracts from GF and GF-conv feces. H) HIF response element (HRE) luciferase assay (HRE-Luc) in HCT116 cells transfected with empty vector (control) or HIF-2α, followed by treatment with vehicle (DMSO) or fecal organic extracts from SPF- (SPF-met) and GF (GF-met) mice (n=3). I) Metabolite screen for HIF-2α suppression based on HRE luciferase assay in HCT116 cells. J) HIF-2α Western analysis in DFO-treated or co-treated with butyrate, propionate and 1,3 diaminopropane (DAP) in HCT116, HT29 and SW480 cells.

All data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (H and I). Western analyses (B, D, E, F, G and J): Images were analyzed by Image J software from three independent experiments, representative image shown. Statistical significance compared with DFO-only or 1% O₂–only (B and E) treatment group. * P < 0.05, *** P < 0.001.