Figure 2. Erastin and RSL3 induce ROS and inhibit prostate cancer cell growth in vitro.

(A, B). Viability (%) of prostate cancer cell lines (DU145, PC3, ARCaP, 22RV1, C4-2, LNCaP and H660) following erastin (0, 1.25, 2.5, 5, 10, and 20 μM) (A) or RSL3 (0, 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 μM) (B) treatment for 72 hours. Experiments were repeated twice independently to verify the reproducibility of the data. Representative experiments are shown. (C, D) ROS measurement by flow cytometry. Cells were treated with erastin (5 μM) (C) or RSL3 (1 μM) (D) for 6 hours followed by incubation with H2DCF for 20 min at 37°C. Relative fluorescence was normalized to untreated control cells and represented as relative percentage of ROS production. Experiments were performed in triplicate and shown. (E, F) Colony formation assay. Prostate cancer cells were grown for nine days in presence of erastin (5 μM) (E) or RSL3 (500 nM) (F). Representative experiments and images are shown. Media containing the indicated compounds was changed every three days. Three independent experiments were performed with triplicate wells. Representative experiments and images are shown. Scale bars represent 4 mm. * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001, ns-no significance, Student’s t-test. Error bars represent mean ± SEM.