E412 01

The collected T24 cells were lysed using radio-immunoprecipitation assay solution (SY4680, YITA Biotechnology, Beijing, China), and protein concentration was examined by bicinchoninic acid kits (P0012, Beyotime). Following separating with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were electrotransferred to polyvinylidene fluoride membranes. Following blockade with 5% skim milk for 2 h, membranes were probed overnight at 4 °C with following rabbit anti-human primary antibodies: matrix metalloproteinase MMP-2 (1/10,000, ab92536, Abcam, Cambridge, UK), light chain 3B (LC3B; 1/50, ab192890), p62 (1/1000, ab207305), phosphorylated p53 (p-p53; 1/1000. ab33889), p53 (1/2000, ab179477), p-mTOR (1/2000, ab109268), mTOR (1/10,000, ab134903), and β-actin (1/1000, ab8227), followed by 1-h incubation with the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (1/50,000, ab205718) at room temperature. Afterward, the images were developed with enhanced chemiluminescence working solution (E412-01, Vazyme). The bands were quantified utilizing Image-Pro Plus 6.0 (Media Cybernetics), with β-actin as an internal reference.

The kidney tissues of mice in each group were added with protein lysate, and the total protein was extracted after homogenization. The protein concentration of each group was calculated by the BCA method and denatured at 5 × loading buffer at 95 °C for 10 min. The total protein loading of each group was 20 μg. Protein samples were electrophoresed in SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The blots were blocked with 5% skimmed milk. Then, they were probed with primary antibodies: GPX4 (ab125066, 1:2000, Abcam, Cambridge Science Park, Cambridge, UK), SLC7A11 (26864-1-AP, 1:1000, Proteintech, Chicago, IL, USA), ACSL4 (ab155282, 1:10,000, Abcam, Cambridge Science Park, Cambridge, UK), TFR1 (bs-21319R, 1:1000, Bioss, Bejing, China), FTH1 (#3998, 1:1000, CST, Boston, MA, USA), FPN1 (2660, 1-1-AP, Proteintech, Chicago, IL, USA), α-sma (# 19245, 1:2000, CST, Boston, MA, USA), and TGF-β1 (#84912, 1:1000, CST, Boston, MA, USA) overnight at 4 °C. The blots were washed and incubated for 1 h at room temperature with the HRP-conjugated secondary antibody and then developed with enhanced chemiluminescence reagents (E412-01, Vazyme, Nangjing, Jiangsu, China). The densitometry of the protein bands was quantified using ImageJ software.

Whole Cell Lysis Assay kit (Keygen, KGP2100) was used to extract proteins from cells and small extracellular vesicles. Total protein concentration was analyzed using the BCA Protein Quantitation Assay kit (Keygen, KGPBCA). Western blotting was performed as previously described [19 (link)]. Antibodies used were as follows: TSG101 (1 : 1000, AF8259, Beyotime), CD63 (1 : 1000, AF1471, Beyotime), CD81 (1 : 1000, AF2428, Beyotime), P21 (1 : 1000, 10355-1-AP, Proteintech), BAX (1 : 1000, 50599-2-Ig, Proteintech), Bcl-2 (1 : 1000, 26593-1-AP, Proteintech), phosphorylated-AKT (p-AKT) (1 : 1000, 28731-1-AP, Proteintech), AKT (1 : 1000, AF1789, Beyotime), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 1000, 10494-1-AP, Proteintech), and beta-actin (β-actin, 1 : 1000, 20536-1-AP, Proteintech). The bands were visualized by using enhanced chemiluminescence (Vazyme, E412-01) and analyzed with a gel documentation system. ImageJ was used for gray analysis of strips.