Fig 1.
ATO upregulates the Nrf2 target genes Hmox-1, Nqo1, and Gclc in wild-type, but not Nrf2-null, splenocytes.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The cells were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for 6 h. Real-time PCR was used to quantify the mRNA expression of (A) Hmox-1, (B) Nqo1, and (C) Gclc. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 2.
ATO markedly inhibits IL-2 production by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The cells were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of IL-2 mRNA by real-time PCR, or (B) 24 h prior to quantification of IL-2 protein in cell supernatants by ELISA. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 3.
ATO markedly inhibits IFNγ production by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of IFNγ mRNA by real-time PCR, or (B) 24 h prior to quantification of IFNγ protein in cell supernatants by ELISA. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 4.
ATO markedly inhibits GM-CSF production by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of GM-CSF mRNA by real-time PCR, or (B) 24 h prior to quantification of GM-CSF protein in cell supernatants by ELISA. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 5.
ATO markedly inhibits protein secretion, but not gene expression, of TNFα by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of TNFα mRNA by real-time PCR, or (B) 24 h prior to quantification of TNFα protein in cell supernatants by ELISA. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 6.
ATO inhibits cytokine production in isolated CD4+ T cells in a Nrf2-independent manner.
Wild-type and Nrf2-null CD4+ T cells were isolated from splenocytes. They were left untreated (BKG) or treated with vehicle (VEH, PBS), 0.1 μM ATO, or 0.5 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for 24 h prior to quantification of (A) IL-2, (B) IFNγ, (C) GM-CSF, and (D) TNFα protein in cell supernatants by ELISA. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 7.
ATO does not affect expression of CD69 or CD25 in anti-CD3/anti-CD28-activated splenocytes.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The cells were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for either (A-B) 6 h prior to quantification of (A) CD25 mRNA and (B) CD69 mRNA by real-time PCR or (C-E) 24 h for quantification of CD25+ and CD69+ cells by flow cytometry. The CD4+ T cells were gated on prior to analysis of CD25 and CD69 expression. (E) Representative dot plots of CD25 and CD69 cell surface protein expression in CD4 T cells. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group. ‡ denotes p<0.05 between the wild-type and Nrf2-null genotypes.
Fig 8.
ATO decreased c-fos DNA binding in anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice.
Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS) or 2 μM ATO for 30 min. The cells were either then left unactivated (BKG) or activated with anti-CD3/anti-CD28 for 4 h after which cells were harvested and nuclear protein extracted. Binding of c-fos to the AP-1 consensus binding site was quantified colorimetrically using an ELISA-based assay. * denotes p<0.05 compared to the wild-type VH group. † denotes p<0.05 compared to the Nrf2-null VH group.