Fig 1: Overexpression of full-length NR4A1 represses SLC30A10 expression and enhances sensitivity to Mn-induced cell death.A, schematic of NR4A1 overexpression constructs. Amino acid numbers and protein domains are indicated. B and C, qRT–PCR in HepG2 cells stably infected with indicated NR4A1 constructs or that underwent control infection (i.e., infection with lentivirus lacking a transfer plasmid) and treated with 0 or 500 μM Mn for 4 h (B) or 500 μM Mn for indicated times (C). Mean expression of control-infected cells without Mn exposure was normalized to 1. N = 9. Mean ± SE. For B, ∗p < 0.05 for indicated comparisons using two-way ANOVA and Tukey's post hoc test. Also for B, there were no differences between control and NR4A11–211 or NR4A1Δ2–251 groups under vehicle or Mn exposure conditions by two-way ANOVA and Tukey’s post hoc test (p > 0.05). For C, ∗p < 0.05 for comparisons between infection conditions at each time point using two-way ANOVA and Sidak’s post hoc test. Data points in the graphs are independent biological replicates. D, cell viability was assayed in HepG2 cells infected as indicated 16 h after treatment with indicated concentrations of Mn. For each infection condition, viability at 0 mM Mn was independently set to 100. N = 3. Mean ± SE. ∗p < 0.05 by two-way ANOVA and Sidak’s post hoc test for differences between infection conditions at each Mn concentration. DBD, DNA-binding domain; LBD, ligand-binding domain; Mn, manganese; qRT–PCR, quantitative RT–PCR; TAD, transactivation domain.
Fig 2: NR4A1 knockdown enhances SLC30A10 expression with or without Mn exposure and protects against Mn-induced cell death in aSLC30A10-dependent manner.A and B, qRT–PCR in wildtype HepG2 cells stably infected with scramble shRNA or shRNA targeting NR4A1 and treated with 500 μM Mn for indicated times. Mean expression at 0 h for scramble shRNA–infected cells was normalized to 1. N = 6. Mean ± SE. ∗p < 0.05 for indicated comparisons using two-way ANOVA and Tukey’s post hoc test. ∗∗p < 0.05 for the comparisons between scramble- or NR4A1-shRNA-infected cells at each time point by two-way ANOVA and Sidak’s post hoc test. C, cell viability in wildtype HepG2 cells infected with scramble shRNA or shRNA targeting NR4A1 16 h after treatment with indicated concentrations of Mn. For each infection condition, viability at 0 mM Mn was independently set to 100. N = 3. Mean ± SE. ∗p < 0.05 by two-way ANOVA and Sidak’s post hoc test for differences between infection conditions at each Mn concentration. D, qRT–PCR in wildtype or ΔSLC30A10 HepG2 cells treated with 250 μM Mn for indicated times. Mean expression in wildtype cells was normalized to 1. N = 4. Mean ± SE. ∗p < 0.05 for comparisons with the 0 h time point within each infection condition using two-way ANOVA and Tukey’s post hoc test. There was also a statistically significant difference between infection conditions (p < 0.05) at the 4 h time point by two-way ANOVA and Sidak’s post hoc test. E, cell viability in ΔSLC30A10 HepG2 cells infected and treated with Mn as described for C. For each infection condition, viability at 0 mM Mn was independently set to 100. N = 3. Mean ± SE. There were no differences between infection conditions at each Mn concentration using two-way ANOVA. Mn, manganese; qRT–PCR, quantitative RT–PCR.
Supplier Page from DNASU for NR4A1 (Homo sapiens) in pDNR-Dual (Creator donor/master vector)