Fig 1: Elevated Mn upregulates NR4A1 expression in aHIF2-dependent manner.A–C, qRT–PCR analyses in HepG2 cells stably infected with scramble shRNA or shRNAs targeting HIF1α and HIF2α and treated with 0 or 500 μM Mn for 4 h. Mean expression in scramble-infected cells without Mn exposure was normalized to 1. N = 9. Mean ± SE. ∗p < 0.05 and ns, not significant for indicated comparisons using two-way ANOVA and Tukey’s post hoc test. D and E, qRT–PCR analyses from previously collected liver samples of endoderm-specific HIF1α or HIF2α knockout mice, with knockout of HIF1α or HIF2α in the liver, or their littermate controls treated with an oral Mn regimen (0 or ∼15 mg Mn/kg daily) from PND 1 and euthanized at PND 14. Mean expression in control genotype without Mn exposure was normalized to 1. N = 5 to 6 per group as indicated. Mean ± SE. ∗p < 0.05 and ns, not significant for indicated comparisons using two-way ANOVA and Sidak’s post hoc test. F, qRT–PCR in HepG2 cells stably infected with VHL-insensitive mouse HIF1α or HIF2α constructs or that were control infected (i.e., infected with lentivirus lacking a transfer plasmid) and treated with 0 or 500 μM Mn for 4 h. Mean expression in control-infected cells without Mn treatment was normalized to 1. N = 9. Mean ± SE. ∗p < 0.05 and ns, not significant for indicated comparisons using two-way ANOVA and Tukey’s post hoc test. G, qRT–PCR in HepG2 cells treated with 0 or 20 μM roxadustat for 16 h. Mean expression in 0 μM roxadustat-treated cells was normalized to 1. N = 9. Mean ± SE. ∗p < 0.05 by t test. Data points in the graphs for all panels are independent biological replicates. Mn, manganese; PND, postnatal day; qRT–PCR, quantitative RT–PCR.