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. 2020 Jun 24;36:101619. doi: 10.1016/j.redox.2020.101619

Fig. 3.

Fig. 3

Mitochondrial translocation of p53 mediated BRD7-enhanced HSC ferroptosis. (A) HSC-LX2 cells with BRD7 knockout and knockin were exposed to erastin (10 μM) for 24 h. Then, the mitochondria, cytoplasm and nucleus were extracted by commercialized kits, respectively. The levels of p53 in cytoplasm, mitochondria, and nucleus were determined by Western blot (n = 3 in every group). (B) HSC-LX2 cells with BRD7 knockin were exposed to erastin (10 μM) for 24 h. The co-localization of p53 (red fluorescence) and mitochondria (mitotracker green) was determined by confocal imaging. Scale bars are 100 μm. Representative photographs were shown (n = 3 in every group). (C) HSC-LX2 cells with BRD7 knockout and knockin were exposed to erastin (10 μM) for 24 h. The expression of total p53 and phosphorylated p53 was detected by Western blot (n = 3 in every group). Serine 392 was substituted by a non-phosphorylatable alanine (S392A mutant) with CRISPR/Cas9 system in HSC-LX2 cells with BRD7 knockin. Then, HSC-LX2 cells were exposed to erastin (10 μM) for 24 h. (D) The levels of p53 in mitochondria were examined by Western blot (n = 3 in every group). (E) Cell viability were determined by CCK8 kit (n = 3 in every group, **P < 0.01, N·S., not significant). (F) The levels of iron, lipid ROS, GSH, and MDA were determined by commercial kits (n = 3 in every group, **P < 0.01, ***P < 0.001). (G) HSC-LX2 cells were transfected with p53 shRNA and BRD7 plasmid, and were exposed to erastin (10 μM) for 24 h. The levels of iron, lipid ROS, GSH, and MDA were determined by commercial kits (n = 3 in every group, **P < 0.01). (H) HSC-LX2 cells were exposed to erastin (10 μM), RSL3 (2.5 μM), and BSO (200 μM) for 24 h. The combination of BRD7 and p53 was examined by immunocoprecipitation (n = 3 in every group). (I) The full-length and different domains of p53 with myc tag were constructed, and then were cotransfected with pcDNA3.1-BRD7 into HSC-LX2 cells. Cell lysates were immunoprecipitated with anti-myc antibody and blotted with anti-BRD7 antibody (n = 3 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)