Ab227256

To further identify the antineoplastic mechanism and performance of AFeC FANDs, the ROS production ability, expressions of FACL4 and GPX4, and the cell disruption of the above-indicated groups were investigated by immunohistochemical and immunofluorescent photomicrographs. For ROS level assay, the mice were intratumorally injected with 10 µL DCFH-DA (10 mM) before photo-excitation. Afterwards, the tumors were excised from the sacrificed mice for immunofluorescent analysis. In addition, the mice with various administration were sacrificed on the 3rd day and the tumors were dissected for staining with FACL4 (Abcam, ab227256, 1:200), GPX4 (Abcam, ab125066, 1:100), or hematoxylin-eosin (H&E), where the FACL4 and GPX4 were used to assess the ferroptosis efficacy, and the H&E stained the nucleus and cytoplasm with blue-purple and red, respectively, to detect the antitumor effect, which were imaged with VS200 research slide scanner (Olympus, Japan).

LUHMES cells in each group were grown on coverslips, fixed with 4% paraformaldehyde for 20 min, and permeated using 0.5% Triton X-100 (Sigma-Aldrich) for 20 min. For the tissue sections, the slices of substantia nigra and corpus striatum were dewaxed with xylene, treated with gradient alcohol, and washed with PBS. Subsequently, the slices were subjected to antigen repair by microwave with citrate. After cooling to room temperature, the slices were treated with 3.3% H₂O₂ at 37°C for 20 min. After washing and blocking, the samples were incubated with anti-ACSL4 (1 : 50 dilution, Abcam, ab227256) and anti-TH (1 : 50 dilution, Abcam, ab137721) overnight at 4°C, then with the corresponding secondary antibody (Abcam) for 1 hour. The nuclei were stained using 10 g/mL DAPI, and the results were immediately photographed under a fluorescence microscope. For details of other experimental methods, please refer to the supplementary materials (available here).

We purchased renal cancer cell lines ACHN from the American Type Culture Collection (Manassas, VA, USA). ACHN cells were cultured in Eagle’s Minimum Essential Medium enriched with 10% Fetal Bovine Serum (FBS) along with 1% penicillin/streptomycin under 37°C along with 5% CO₂ conditions.
G-CLONE supplied the small chemicals sorafenib (GS0220) along with sodium nitroprusside (GN0200).
Sigma-Aldrich provided us with rapamycin (V900930). ABCAM supplied antibodies to ACSL4 (ab227256), beta-tubulin (ab6046), Beclin-1 (ab210498), LC3 (ab192890), FTH (ab183781), ATG12 (ab155589), and NCOA4 (ab86707). Secondary antibodies linked to HRP, as well as chemiluminescent reagents, were bought from Santa Cruz Biotech (Dallas, TX, USA).
Initially, all chemicals were dispersed in dimethylsulfoxide (DMSO) as a stock solution. The figure legends indicated the chemical treatment duration along with the final levels