In vitro activity: Ferrostatin-1 (2 μM) prevents erastin-induced ferroptosis in cancer cells, as well as glutamate-induced cell death in postnatal rat brain slices. Ferrostatin-1 is a lipid ROS scavenger, with the N-cyclohexyl moiety serving as a lipo-philic anchor within biological membranes. Ferrostatin-1 does not inhibit extracellular signal -regulated kinase (ERK) phos-phorylation or arrest the proliferation of HT-1080 cells, suggesting that it does not inhibit the MEK/ERK pathway, chelate iron, or inhibit protein synthesis. Ferrostatin-1 does, however, prevent erastin-induced accumulation of cytosolic and lipid ROS. Ferrostatin-1 readily oxidizes the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) under cell-free conditions.

Kinase Assay: The cells treated for 24 h with Ferrostatin-1 are washed three times with phosphate-buffered saline (PBS) and pelleted by centrifugation. The supernatant is removed and 80 μL of lysis buffer is added to the cells and then stored overnight at -20°C. Subsequently, the cells are centrifuged at 10,000 RPM for 12 min and both the pellet and supernatant are stored for future use.

Cell Assay: BJ-TERT/LT/ST/RASV12 cells are seeded in 100 mm dishes and allowed to grow overnight. Cells are treated with erastin (5 or 10 μg/ml) for 6, 8, or 11 hr. A camptothecin-treated (0.4 μg/ml) control is maintained, treated at the time of seeding for 20 hours. After the treatment, cells are harvested with trypsin/EDTA and washed once with fresh medium containing serum and then twice with phosphate-buffered saline. Cells are resuspended in 1× binding buffer. 100 μL is incubated with 5 μL of Annexin V-FITC and propidium iodiode for 15 min in the dark at room temperature. Then 400 μl of the 1× binding buffer s added and the cells analyzed by flow cytometry. Data are acquired and analyzed using Cellquest software. Only viable cells that do not stain with propidium iodiode are analzyed for Annexin V-FITC staining using the FL1 channel.