Gpx4 67763 1 ig

The antibodies used targeted the following proteins (dilutions used are included):
GAPDH (CW0101: immunoblotting, 1:1000) from CWBIOTECH; F-actin (40734ES75: immunofluorescence, 1:100) from YEASEN; U1A (10212: immunoblotting, 1:500) from Proteintech; CD63 (67605-1-Ig: immunoblotting, 1:5000) from Proteintech; CD9 (60232-1-Ig: immunoblotting, 1:5000) from Proteintech; TSG101 (14497-1-AP: immunoblotting, 1:1000) from Proteintech; GPX4 (67763-1-Ig: immunoblotting, 1:5000; IHC, 1:1000) from Proteintech; and DHODH (14877-1-AP: immunoblotting, 1:2000; IHC, 1:100) from Proteintech.
The inhibitors used are as follows:
Sorafenib (HY-10201: 10 μM for the in vitro assay and 30 mg/kg for the in vivo assay) and Ferrostatin-1 (HY-100579: 60 nM for the in vitro assay), and Ferrostatin-1 (HY-100579: 5 mg/kg, intraperitoneal injection for the in vivo assay) were obtained from MedChem Express.

Tissue specimens from human or mouse were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned with 6-μm thickness, and immunostained with specific antibodies, including SND1 (10,760–1-AP, Proteintech), Nrf2 (16,396–1-AP, Proteintech), GPX4 (67,763–1-Ig, Proteintech), and 4-HNE (R&D systems, MAB3249). The histological slides were observed under a light microscope (Leica, Germany). The percentage of positive cells was calculated.
TUNEL staining was performed with In Situ Cell Death Detection Kit, POD (Roche, Switzerland) according to the manufacturer’s protocol. Images were acquired with an Olympus FSX100 microscope (Olympus, Tokyo, Japan).

Dulbecco's modified Eagle's medium; high glucose; L-glutamine, phenol red (DMEM, 11965092); fetal bovine serum (FBS, 10270106); penicillin/streptomycin (10378016); PBS (10010023); trypsin-EDTA; 0.05% phenol red (25200072); and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT, V13154) were supplied by Gibco (NY, USA). Giemsa staining solution (C0133) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). N-acetyl-cysteine (NAC, A9165), 3-methyladenine (3-MA, M9281), ferrostatin-1 (Fer-1, SML0583), Z-VAD-FMK (Z-VAD, V116), necrostatin-1 (Nec-1, N9037), and belnacasan (VX-765, 5313720001) were purchased from Sigma-Aldrich (Darmstadt, Germany). Primary antibodies against p53 (60283-2-Ig), p62 (66184-1-Ig), Beclin1 (11306-1-AP), xCT/SLC7A11 (26864-1-AP), and GPX4 (67763-1-Ig) were purchased from Proteintech (Wuhan, China). Primary antibodies against Atg7 (8558T), LC3A/B (12741S), KEAP1 (8047S), NRF2 (12721T), NCOA4 (66849S), FTH1 (4393S), DMT1/SLC11A2 (15083S), and GAPDH (5174S) and rabbit (7074P2) or mouse (7076P2) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). For in vitro experiments, Rh4 was dissolved in DMSO and medium, and the final concentration of DMSO in medium was ≤0.3% (vol/vol).