0.8 million HT-1080 cells were seeded per well in a 60 mm plate and allowed to adhere overnight. Cells were then co-treated with 100 μM α-Tocopherol and either 1 μM of RSL3, 10 μM of Erastin, 5 μM of FIN56, 10 μM of FINO2, or vehicle for 10 h. Cells were harvested with trypsin (Invitrogen, #25200-114), pelleted, and frozen at -80˚C. Cell pellets were thawed, lysed, blotted, and imaged as previously described.3 (link) In particular, for this experiment, the set of 15 pellets was run on a single gel. Ultimately, two gels were run and quantified from a total of six biological replicates (30 pellets in total). For the other proteins of interest, experiments were performed in biological triplicate. Antibodies used were: GPX4 (abcam, #ab125066, 1:250 dilution), Ferritin Light Chain (Santa Cruz, #sc-390558, 1:1000 dilution), IRP2 (Nous Biological, #NB100-1798, 1:1000 dilution), Transferrin receptor 1 (CD71, TFRC, Cell Signaling, #13113, 1:1000 dilution), Actin (Cell signaling, #D18C11, 1:3000 dilution), alpha-tubulin (Santa Cruz, #sc-32293, 1:3000 dilution) and GAPDH (Santa Cruz, # sc-47724, 1:10,000 dilution). Results were quantified using a LI-COR Odyssey CLx IR scanner and GraphPad Prism 7.