Fig 2: NRF2 agonist prevention of UVB-induced skin ear pigmentation in mice is specific to NRF2 signaling and requires keratinocyte-intrinsic IL-6Ra signaling.(A) Representative indirect immunofluorescence for IL-6Ra. DAPI, nuclear staining; epi, epidermis; derm, dermis. Dotted lines delineate the dermoepidermal junction. Scale bar: 50 µm. (B) Fold change of immunofluorescence signal for IL-6Ra (mean ± SEM). (C) Schematic of preventative treatment regimen for K14-Cre-ERT2IL-6Rafl/fl mice that were either untreated (Un) or received UVB exposure alone (UVB), UVB + vehicle treatment (UVB+OIL), or UVB + NRF2 agonist (SF) treatment (UVB+SF). (D) Representative images of ear skin. (E) Fold change of skin darkness (mean ± SEM). (F) Representative indirect immunofluorescence for NRF2. Scale bar: 50 µm. (G) Proposed mechanism. *P < 0.05, between indicated groups as calculated by a Mann-Whitney U test. P values were corrected for multiple comparisons using a Bonferroni correction.

Fig 3: Nrf2 overexpression suppressed pyroptosis in the cellular PD model. (A) Proliferation rates of Nrf2-overexpressing PC12 cells exposed to 6-OHDA. Cell proliferation was assessed by MTS assay. (B) Alterations in apoptotic PC12 cells overexpressing Nrf2 following 6-OHDA exposure. Apoptosis was determined by flow cytometry. (C) Differential expression of Nrf2 and pyroptosis marker proteins in Nrf2-overexpressing PC12 cells exposed to 6-OHDA. Protein levels in PC12 cells were analyzed by Western blotting. (D) Relative protein expression was counted in Western blot. (E and F) Changes of IL-1ß and LDH release in the cellular PD model with Nrf2 overexpression. Marker protein contents were detected by Western blotting. *P < 0.05; **P < 0.01.

Fig 4: Nrf2 prevents critical oxidative damage in DS cells. (a) Inhibition of pNRF2 translocation decreases DS cell proliferation. NL and DS cells were treated with 2 nM Gö6983 or vehicle for 36 hr and then nuclei were counted. (b) Inhibition of pNRF2 translocation increases ROS generation. DS cultures expressing EV or mCAT were treated with 2 nM Gö6983 or vehicle for 12 hr and stained with DCF to measure ROS levels. Cells treated with 2 nM Gö6976 were included as a control. Scale bar = 20 µm. *p < 0.05. (c) Proliferation was evaluated by BrdU incorporation in NL or DS cultures expressing EV or mCAT. Proliferating cells (BrdU +) over total number of cells (DAPI +). *p < 0.05. (d) Increased cell area in DS fibroblasts. NL and DS cells expressing EV or mCAT vector were seeded at low density, grown for 24 hr, fixed and stained with Alexa-546 Phalloidin and DAPI to visualize cellular an nuclear areas, respectively. Scale bar = 20 µm. * p < 0.05. a and c: 6 culture replicates (four 10× fields each) analyzed per condition; b: at least 20 cells included in the analysis per condition; d: 75–80 cells per condition

Fig 5: Activation of the Nrf2 signaling pathway in DS and Dp16 cells. (a) WB analysis of total NRF2 levels in NL and DS cultures infected with EV or mCAT. Samples were probed against total NRF2 and CAT. ATP5a and Tub protein levels were used as loading controls. (b) Immunofluorescence against active pNRF2ser40 (green) performed in NL and DS cultures expressing EV or mCAT. Nuclei were stained with DAPI. pNRF2 nuclear localization were considered positive when nuclear over cytoplasmic fluorescence intensity ratio was greater than 1. White arrow: no translocation; Red arrowheads: NRF2-positive nuclei. Scale bar = 20 µm. (c) Total NRF2 levels were visualized by western blot of WT and DP16 cultures expressing EV or mCAT. Note the marked reduction of Nrf2 levels in Dp16 fibroblasts expressing mCAT. (d) PKCd protein levels by wb in NL and DS fibroblasts infected with EV or mCAT. PKCd expression did not change on analyzed samples. (e) Increased pNRF2 nuclear translocation in DS cells. PKCd is activated by caspase 3, and then phosphorylates Nrf2. PKCd regulation of NRF2 activity was determined by PKCd inhibitor Gö6983 (2 nM). Gö6976 (2 nM), a specific inhibitor of PKCa and PKCß, did not affect Nrf2 translocation. Casp3 dependent PKCd activation was tested by Z-DEVD-FMK (Z-DEVD-F) Casp3 inhibitor (2 nM). DS cultures expressing EV or mCAT were treated with 200 µM PQ for 48 hr to further increase pNRFser40 nuclear localization and then incubated for 12 hr with inhibitors. *p < 0.05. b and e: 50–60 cells were included in the analysis, for each experimental condition