Gpx 4

Total protein was extracted from the tumor tissue using RIPA protein extraction reagent containing phosphatase and protease inhibitors. The total protein concentration was measured using a BCA Protein Assay Kit. Then, the protein samples were mixed with a loading buffer (containing β-mercaptoethanol) and were heated at 100 ℃ for 10 min. Immunoblotting was performed using Bcl2-Associated X Protein (BAX) (Abcam, ab32503), B-cell lymphoma 2 (Bcl-2) (Abcam, 182858), SLC7A11 (Huabio, HA600097), GPX-4 (Bioss, 3884R), p53 (Proteintech, 60283-2-1g), GSK-3β (Huabio, ET1607-71), p-GSK-3β (Bioss, 2066R), Nrf-2 (Santa Cruz, 365949), cluster of differentiation 31 (CD31) (Huabio, ER31219), vascular endothelial growth factor-A (VEGFA) (Huabio, ET1604-28), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), Histone-H3 (Proteintech, 17168-1-AP), and β-actin (Proteintech, HRP-60008). Densitometry was performed using ImageJ software.

Western blotting was performed as previously described [30 (link)]. The antibodies used were as follows: GPX4 (#bs-3884 R, Bioss, Beijing, China), IREB2 (#bs-4484 R Bioss), GAPDH (#KC-5G5, Aksomics, Shanghai, China), HRP-conjugated secondary antibodies to rabbit (#4050-05, Southern biotech, AL, USA). Original images of WB were provided in Supplementary.

Kidney tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sliced into 5μm sections based on routine protocols. Immunohistochemical (IHC) and immunofluorescence (IF) were performed as previously described [27 (link),28 (link)]. The positive area of CAT (Bioss; 1:100) and FSP1 (Proteintech; 1:50), and the fluorescence intensities of GPX4 (Bioss; 1:100), (Abclonal; 1:50), Nrf2 (Bioss; 1:100) and HO-1 (Proteintech; 1:50) in the photos were detected by Image J software.