Df6278

Liver tissues or harvested cells, as indicated, were lysed with RIPA buffer complemented with protease inhibitors. The protein concentration was evaluated through the BCA protein assay kit. In brief, a total of 30 μg proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes. Primary antibodies used were anti-xCT (ab37185, Abcam, 1:1000), anti-GPx4 (ab125066, Abcam, 1:1000), anti-ACSL4 (DF12141, Affinity, 1:1000), anti-p53 (2524, CST, 1:1000), anti-FTH1 (DF6278, Affinity, 1:1000), anti-Nrf2 (12721, CST, 1:1000), anti-LaminB (13435, CST, 1:1000), anti-β-actin (3700, CST, 1:1000), and anti-GAPDH (abs100005, Absin, 1:1000). Second antibodies were peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (1:5000) (Zhongshan Golden Bridge Biotechnology, Beijing, China). To visualize the proteomic bands, an enhanced western luminescent detection kit (Vigorous Biotechnology, Beijing, China) was employed. The densitometry results were quantitatively analyzed by using the Image J software, with β-actin, GAPDH, or LaminB bands being normalized/internal controls as appropriate.

RNA levels were measured by qPCR analysis following to the manufacturer’s instructions. Western blot analysis was also performed according to the manufacturer’s protocol. Briefly, cells or tissues were lysed in RIPA buffer. The protein concentrations were then normalized using a BCA assay kit (Solarbio, Beijing, China). Anti-GAPDH (1:1000, ABclonal, Wuhan, China, AC001), anti-STK33 (1:1000, Abcam, Cambridge, MA, USA, ab206296), anti-MDR1 (1:1000, Affinity, Jiangshu, China, AF5185), anti-GSS (1:1000, Affinity, DF6214), anti-MDM4 (1:1000, Affinity, DF8676), anti-P450 3A4/5 (1:1000, Affinity, AF5312), anti-E-cadherin (1:1000, Affinity, AF0131), anti-SLC7A11 (1:1000, Affinity, DF12509), anti-GPX4 (1:1000, Affinity, DF6701), anti-FTH1 (1:1000, Affinity, DF6278), anti-FSP1 (1:1000, Affinity, DF6516), anti-KRas (1:1000, Affinity, DF6324), and anti-ubiquitin antibodies (1:1000, Affinity, AF0289) were used in this study.

Immunofluorescence staining was performed as previously described [35 (link)]. Frozen brain sections (7 μm) or cultured cells on coverslips were fixed in 4% paraformaldehyde for 10 min. Then, they were treated with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime, Shanghai, China) for 30 min and were blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 60 min. These samples were incubated with specific primary antibodies for ACSL4 (Affinity, DF12141, 1 : 200), FTH (Affinity, DF6278,1 : 200), GPX4 (Affinity, DF6701,1 : 200), NeuN (MilliporeSigma, MAB377X, 1 : 200), 8-hydroxyguanosine (8-OHdG) (Bioss, bs-1278R, 1 : 100), and SIRT1 (Santa Cruz, sc-74465, 1 : 50) in Universal Antibody Diluent (New Cell & Molecular, Suzhou, China) at 4°C overnight. Then, the samples were slowly washed three times with phosphate-buffered saline with 0.5% Tween-20 (PBST) and incubated with corresponding secondary antibodies (Cy™3-conjugated goat antirabbit IgG or goat antimouse IgG, 1 : 200) for 1 h at room temperature (RT). After washing 3 times with PBST, the samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma, 1 : 2000) for 10 min at RT. Fluorescence was visualized by a fluorescence microscope (ZEISS, Scope A1, Germany).