Lip 1

A total of 5 × 10⁶ SW620 cells in 150 μL PBS was subcutaneously injected into the mouse dorsal flanks. One week after inoculation, the tumor size was measured along two perpendicular axes every other day using a vernier caliper. Three weeks after inoculation, tumors were isolated, weighted, and documented after euthanizing the mice. The formula (volume = length × width² × 0.5) was used to calculate the volume of tumors. For the in vivo drug administration experiment, the mice were randomly divided into groups. ML162 (TOPSCIENCE, Cat#T8970) and Liproxstatin-1 (Lip-1) (TOPSCIENCE, Cat#T2376) were dissolved in vehicle (5% DMSO, 30% PEG300, 10% Tween-80 in saline). ML162 and Lip-1 were intraperitoneally injected at a dose of 10 mg/kg every day since the 4th day of tumor cell inoculation, and the vehicle was used as control. The mice were euthanized after two weeks of drug administration, and the subcutaneous tumors were isolated for further analysis.

Bavachin was purchased from MCE (China), and pifithrin-α (PFT-α), deferoxamine (DFO), ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1) were purchased from Topscience (China). Vitamin E was purchased from Beyotime (Shanghai, China). Rabbit polyclonal anti-transferrin receptor antibody (TFRC, AF5343, 1 : 1000), rabbit polyclonal anti-divalent metal transporter-1 antibody (DMT1, DF12740. 1 : 1000), rabbit polyclonal anti-ferritin light chain antibody (FTL, DF6604, 1 : 1000), rabbit polyclonal anti-ferritin heavy chain antibody (FTH, DF6278, 1 : 1000), rabbit polyclonal anti-SLC7A11 antibody (DF12509, 1 : 1000), rabbit polyclonal anti-P53 antibody (AF0879, 1 : 1000), rabbit polyclonal anti-STAT3 antibody (AF6294, 1 : 1000), rabbit polyclonal anti-p-STAT3 (705) antibody (AF3293, 1 : 1000), and rabbit polyclonal anti-glutathione peroxidase-4 antibody (GPX4, DF6701, 1 : 1000) were purchased from Affinity Bioscience (China). HRP goat anti-rabbit IgG was purchased from Earthox (USA).