Fig 2: NRF2 specifically regulates NADK Isoform 3(A) Schematic of the sequence of the ARE in the Isoform 3 promoter. mRNA levels of Isoform 3 in A375 melanoma cells with ARE deletion.(B) mRNA levels of NRF2 targets NQO-1 and HO-1 in PDX tumor M481 (left) and M405 (right) in subcutaneous tumor compared to metastatic nodules. Expression levels of NRF2 PDX tumor M481 after nuclear fractionation.(C) ChIP seq data looking at Pol2 and activating histone marks.(D) NADK Isoform 1 and 3 mRNA expression in A375 melanoma cell line treated with Ki676 (5uM) for 30 min(E) NADK Isoform 1 and 3 mRNA expression in A375 melanoma cell line NRF2 knock down treated with Ki676 (5uM) for 30 min(F) GFP mRNA levels in A375 melanoma cell line NRF2 knock down expressing NADK Isoform 1 or Isoform 3 reporter and treated with pyocyanin (10uM) for 24 h.

Fig 3: NF-κB signaling is required for NBCn1 activation. (A) Protein expression levels of NRF2 in A549 and NBCn1overexpressing HEK293T [NBCn1 (H.T)] cells. β-actin was used as a loading control. (B) Relative intensity of NRF2 normalized to β-actin. Data are presented as mean ± SEM (n = 4, *** p < 0.001 vs. A549). (C) Protein expression levels of total NF-κB and p-NF-κB in NBCn1 (H.T) cells treated with H2O2 (10 μM, 1 h) with or without S0859 (S; 40 μM, 1 h). β-actin was used as a loading control. (D) Relative intensity of p-NF-κB normalized to total NF-κB. Data are presented as mean ± SEM (n = 3, ** p < 0.01 vs. control; ## p < 0.01 vs. H2O2). (E) BCECF-based intracellular pH traces of NBC activity in NBCn1 (H.T) cells treated with H2O2 with or without JSH-23 (20 μM, 1 h). (F) NBC activity was assessed by measuring the recovery rate of pHi (ΔpH/sec). Data are presented as mean ± SEM (n = 4, * p < 0.05 vs. control; # p < 0.05 vs. H2O2). (G) Protein expression levels of NBCn1 in NBCn1 (H.T) cells treated with H2O2 with or without S0859. β-actin was used as a loading control. (H) Relative intensity of NBCn1 normalized to that of β-actin. Data are presented as mean ± SEM (n = 7, * p < 0.05 vs. control; ## p < 0.01 vs. H2O2). (I) BCECF-based intracellular pH traces of NBC activity in NBCn1 (H.T) cells treated with H2O2 in the presence or absence of S0859. (J) NBC activity was assessed by measuring the recovery rate of pHi (ΔpH/sec). Data are presented as mean ± SEM (n = 4, * p < 0.05 vs. control; # p < 0.05 vs. H2O2). (K) Representative images of transwell migration assays in NBCn1 (H.T) cells treated with H2O2 with or without S0859. Cells were stained with DAPI. Scale bar = 200 μm. (L) Analysis of cellular migration is shown as a relative migration ratio. Data are presented as mean ± SEM (n = 4, * p < 0.05 vs. control; ## p < 0.01 vs. H2O2).

Fig 4: Nrf2 expression in activated mouse, human and Jurkat T cells(A) Gating strategy used for activated mouse T cells showing Nrf2 expression at 0 h. Cells were gated for singlets (FSC-A vs FSC-H), live cells (FSC-A vs viability dye), FSC-A vs SSC-A, then CD3+ T cells, followed by gating on CD4+ and CD8+ T cells.(B) Representative histogram overlays and summary graphs showing Nrf2 expression in activated mouse CD4+ and CD8+ T cells (n=4).(C) Gating strategy for activated human T cells showing Nrf2 expression at 0 h.(D) Representative histograms overlays and summary graphs showing Nrf2 expression in activated human CD4+ and CD8+ T cells (n=4).(E) Gating strategy for activated Jurkat T cells showing Nrf2 expression at 0 h.(F) Representative histograms overlays and summary graphs showing Nrf2 expression in activated Jurkat T cells (n=2 technical duplicates). All data are shown as mean ± SEM from the indicated numbers of mice. ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.