In vitro activity: Erastin is selectively lethal to oncogenic RAS-mutant cell lines, and triggers a unique iron-dependent form of non-apoptotic cell death called ferroptosis. Erastin binds directly to VDAC2 and causes mitochondrial damage via ROS production in an NADH-dependent manner, which induces cell death in some tumor cells harbouring activating mutations in the RAS-RAF-MEK pathway. In addition, erastin, via inducing ROS-mediated CID (Caspase-independent cell death), strongly enhances the effect of cisplatin in WT EGFR cells

Kinase Assay: To test erastin’s activity on colorectal cancer cell survival, HT-29 cells are treated with increasing concentrations of erastin (0.1–30 μM). MTT assay was performed.

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.