Fig 1: NR4A1 is aMn-responsive gene that represses SLC30A10 expression.A–F, qRT–PCR analyses in HepG2 cells stably infected with scramble shRNA or shRNA targeting NR4A1 (A & B), p53 (C & D), or PPARA (E & F). For each panel, mean expression in scramble-infected cells was normalized to 1. N = 3. Mean ± SE. ∗p < 0.05 by t test. Data points in the graphs are independent biological replicates. G and H, qRT–PCR in HepG2 cells treated with 500 μM Mn for indicated times (G) or indicated concentrations of Mn for 8 h (H). Mean expression without Mn exposure (0 h for G or 0 μM Mn for H) was normalized to 1. N = 3 for G and 4 for H. Mean ± SE. ∗p < 0.05 for difference with the no Mn condition for each gene separately by one-way ANOVA and Dunnett’s post hoc test. For H, the extracellular Mn EC50 was calculated using nonlinear regression and log(agonist) versus response (three parameters) with the bottom constrained to 1. I, qRT–PCR in differentiated SH-SY5Y cells treated with 500 μM Mn for indicated times. For each gene, mean expression at 0 h was normalized to 1. N = 9. Mean ± SE. ∗p < 0.05 for difference between 0 h and other time points for each gene separately by one-way ANOVA and Dunnett’s post hoc test. J, qRT–PCR in HMC3 cells treated with 500 μM Mn for indicated times. Mean expression at 0 h was normalized to 1. N = 6. Mean ± SE. ∗p < 0.05 for difference with the 0 h time point by one-way ANOVA and Dunnett’s post hoc test. Mn, manganese; qRT–PCR, quantitative RT–PCR.