Fig 2: The expression of KEAP1 and PGAM5 were upregulated in melanocytes under oxidative stress.The location of KEAP1 (A) and PGAM5 (B) in PIG1 cells stimulated by H2O2 or pretreated with Z-VAD and Nec-1 prior to H2O2 disposition, detected by immunofluorescence assay. C The expression of KEAP1 and PGAM5 in PIG1 cells treated with H2O2 or pretreated with Z-VAD and Nec-1 prior to H2O2 disposition, detected by Western-blot assay. ***P < 0.001, ns: not significant.

Fig 3: KEAP1 and PGAM5 participate in the cell death of melanocytes under oxidative stress.The cell viability of PIG1 cells treated by H2O2 or pretreated with KEAP1 siRNA prior to H2O2 disposition is determined by CCK8 assay (A) and Resazurin-conversion assay (B). C The activity of released LDH in PIG1 cells treated by H2O2 or pretreated with KEAP1 siRNA prior to H2O2 disposition, determined by LDH release assay. D Annexin V-FITC/PI apoptosis assay of PIG1 cells pretreated with KEAP1 siRNA, followed by treatment with H2O2. The cell viability of PIG1 cells treated by H2O2 or pretreated with PGAM5 siRNA prior to H2O2 disposition is determined by CCK8 assay (E) and Resazurin-conversion assay (F). G The activity of released LDH in PIG1 cells treated by H2O2 or pretreated with PGAM5 siRNA prior to H2O2 disposition, determined by LDH release assay. H Annexin V-FITC/PI apoptosis assay of PIG1 cells pretreated with PGAM5 siRNA, followed by treatment with H2O2. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: KEAP1 is involved in NRF2 pathway activation in TTM-treated HUVECs.A Western blot analysis and quantification of KEAP1 in 0, 10, 30, 100, or 300 µM TTM-treated HUVECs. ß-Actin was used as loading control. B Western blot analysis and quantification of KEAP1 and GAPDH in HUVECs treated with TTM (100 µM) for 0, 3, 6, 9, and 12 h, respectively. C qPCR analysis mRNA levels of HMOX1, GCLM, SLC7A11 in KEAP1-OE cells treated with or without TTM. One-way ANOVA followed by a Tukey multiple comparison test was used for statistical analysis. *p < 0.05. D Western blot analysis and quantification of KEAP1, NRF2, HMOX1, and GCLM in KEAP1-OE cells treated with or without TTM. ß-Actin was used as loading control. E Plasmid vectors expressing KEAP1-Flag (WT), KEAP1-Flag (C151S) or KEAP1-Flag (C151A) were co-transfected with NRF2-Myc into Hela cells for 48 h and then treated with or without 100 µM TTM for 12 h. Western blot analysis and quantification of protein levels of exogenous NRF2-Myc, KEAP1-Flag and endogenous KEAP1. ß-Actin was used as loading control.

Fig 5: KEAP1 disruption confers resistance to lenvatinib.Cells were treated with the indicated concentrations of lenvatinib for 48 hours. (A) Cell viability was assessed using the MTS assay. The results were derived from three independent experiments. (B) ROS levels were measured as indicated in the Materials and Methods section. The results were derived from seven independent experiments.