Extended Data Fig. 3: Tumoral hepcidin regulation in vitro and in vivo.

qPCR analysis for hepcidin (Hamp) after treatment with FG4592 (100 μM) or vehicle for 16 hours in (a) HCT116 cells (N = 3 biologically independent cell replicates) and (b) enteroids generated from mice with inducible, colon epithelial deletion of APC and p53 and activation of KRAS (N = 3 biologically independent samples). c, qPCR analysis of Hamp in the colon of mice with embryonic, intestinal epithelial-specific overexpression of HIF-2α (HIF-2α^OE) compared to wild-type mice (HIF-2α^WT) (HIF-2α^WT N = 6 and HIF-2α^OE N = 10 biologically independent samples from independent animals). d, qPCR analysis of Hamp in the colon of colon epithelial-specific HIF-2α^WT and HIF-2α^OE mice that are also deficient for APC for 30 days HIF-2α^WT N = 3 and HIF-2α^OE N = 3 biologically independent samples from independent animals). e, qPCR analysis of Hamp in HCT116 cells treated for 24 hours with conditioned media (CM) from RAW 264.7 macrophages that had been treated with 10 ng/mL LPS for 16 hours (N = 3 biologically independent cell replicates). f, Relative luciferase activity of the human hepcidin promoter in HCT116 cells treated with vehicle (Veh), live bacteria (Live), or heat-killed bacteria (HK), or (g) vehicle (Veh) or bacteria-derived metabolites (N = 3 independent cell replicates). h, Methylation status of the human hepcidin promoter in human colorectal cancer tissue (Normal N = 37 and Tumor N = 313). i, HCT116 cells treated with vehicle (Veh) or 5AZA (10 μM) for 72 hours and then treated with vehicle (Veh) and/or FG4592 (100 μM) for 16 hours and analyzed via Western blot analysis for DNA methyltransferase 1 (DNMT1) and (j) qPCR analysis for HAMP expression (N = 3 biologically independent cell replicates). k, HCT116 cells pretreated with or without FG4592 (100 μM) for 16 hours and then administered a panel of epigenetic modifiers for 24 hours. qPCR was used to measure the expression of HAMP and the HIF-2α target, ANKRD37 (N = 3 biologically independent cell replicates for each treatment). l, Patient-derived enteroids were either pretreated with or without FG4592 (100 μM) for 16 hours and were then administered a panel of epigenetic modifiers for 24 hours. qPCR was used to measure the expression of HAMP and the HIF-2α target, ANKRD37 (N = 3 biologically independent samples for each treatment). Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired t test (a-e, h) or 1-way ANOVA with Tukey’s post hoc (f, j, k-l). ****P < 0.0001.

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