Compound 4 is a Type I inhibitor. (A) 4 depletes reduced thiols. NCI-H522 cells were exposed to either 4 or erastin. Thiols were measured 6 hours later using monochlorobimane. (B) Oxidation of a glutathione biosensor in cells exposed to compound 4. HT1080 cells were transiently transfected with Grx1-roGFP2, exposed to 20 μM compound 4 for 8 hours and subject to live-cell confocal imaging. Examples of scanned cells are shown in (B), with pixel intensities of representative cells shown in (C). Excitation and emission settings are shown in the figure. In (D), the ratio of pixel intensities excited at 405 nm versus 488 nm were compiled for at least 45 cells. (E) 4-induced cell death is blocked by N-acetylcysteine (NAC). NCI-H522 cells were exposed to the indicated compounds for three days. Viability was determined by methylene blue staining. (F) 4 inhibits glutamate release. HT1080 cells exposed to the compounds indicated were incubated in medium lacking glutamate for up to 8 hours. Conditioned media were assayed for secreted glutamate. At 8 hrs, 5 μM and 10 μM compound 4 were significantly lower than DMSO (p value of 0.01 and 0.002 respectively). (G) 4 inhibits Cystine-FITC uptake. MDA-MB-231 cells were exposed to 4 with or without liproxstatin (2.5 μM) for 16 hours. Cystine-FITC was added at 5 μM and images collected 1 hour later. Pixel intensities from digital images of at least 70 cells are compiled in (H).