Fig. 5

MMA inhibits NRF2 nuclear translocation. In vitro, AC16 cells were treated with MMA (17.5 mM) alone for 3 h or with (acetylcysteine) NAC (5 mM) for 2 h followed by MMA (17.5 mM) for 3 h. a-d NRF2 expression in the cytoplasm, nucleus and total protein of AC16 cells analysed by WB (n = 3/group). e, f Immunofluorescence analysis of the effect of MMA and NAC on NRF2 nucleus translocation in AC16 cells (n = 3/group) (Scale bar: 50 μm). g, h Binding model between KEAP1 and NRF2 were acquired by protein–protein predicted website BioGRID (http://thebiogrid.org) and HDOCK SEVER (http://hdock.phys.hust.edu.cn/). i, j Co-immunoprecipitation detected NRF2 and KEAP1 complex formation (n = 3/group). k-l The expression of ferroptosis-related proteins GPX4 and SLC7A11 were detected by western blot (n = 3/group). m Western blotting analysis of the protein level of NRF2 in AC16 cells in the nucleus (n = 3/group). Data are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and n.s, not significant