Fig. 4

P53-NRF2 interaction and p53-mediated suppression of NRF2 activity are diversely regulated by p62 according to p53 status. A The localization of p53 and NRF2 was detected by immunofluorescence in U251 and A172 cells. Scar bars = 10 um. B Co-immunoprecipitation assays were performed to assess the association between NRF2 and p53. 293 T cells were co-transfected with HA-NRF2 and Flag-WT-p53 plasmids. Lysates of 293 T cells were subjected to IP using anti-Flag or anti-HA antibodies, followed by immunoblotting with anti-Flag and anti-HA antibodies. Non-specific IgG was used as a control. Whole cell lysates were used as an input control. C U251 cells were transfected with pcDNA3.1 or Flag-R273h-p53 plasmids, followed by erastin treatment or not. A172 cells were transfected with pcDNA3.1 or Flag-WT-p53 plasmids, followed by erastin treatment or not. The protein expression of SLC7A11, NRF2, p53, p62, HO-1 and NOQ-1 were detected by western blot analysis. D The quantification in Fig. 4C. E The overexpression of R273h mutant p53 inhibited the NRF2-driven luciferase activity in U251 cells by dual luciferase reporter assays. The overexpression of wild-type p53 inhibited the NRF2-driven luciferase activity in A172 and U87 cells by dual luciferase reporter assays. Cells were treated with DMSO or Erastin. The luciferase activity was calculated as Firefly luciferase/Renilla luciferase. F The effect of p62 overexpression on NRF2-driven luciferase activity in U251, U87 and A172 cells were determined by dual luciferase reporter assays. Cells were treated with DMSO or Erastin. G The effect of p62 overexpression on p53-NRF2 association in different p53 status was determined by IP analysis