Figure 4.

Effects of BMS536924 on insulin signaling or the ERK pathway does not explain inhibition of ferroptosis. (A, B and C). Cells were exposed to compound 1 in the presence or absence of IGF1 and viability determined by methylene blue staining. (A) NCI H522 and (B) MDA MB 231 cells were treated with compound 1 (as indicated) and/or IGF1 (100 ng/ml) and/or BMS536924 (0.5 μM), 3 days later cell viability was measured. (C) MDA MB 231 cells were starved for 24 hours (DMEM without serum) and then exposed to compound 1 in the presence or absence of IGF1 before analysis of viability. (D) IGF1 induces and BMS536924 blocks Akt1 phosphorylation. MDA MB 231 cells were starved (DMEM without serum) or incubated in DMEM plus 10% FBS for 24 hours. Next, cells were treated with IGF1 (100ng/ml) or BMS536924 (1μM) for 8 or 24 hours. Western blot was probed for p-Akt1, total Akt1 and actin, as a loading control. Akt phosphorylation was measured as a marker for activation of the insulin receptors. (E & F) Effects of BMS536924 on ERK phosphorylation. HT1080 cells were exposed to either BMS536924 or DFO for 24 hours and then analyzed by western blotting with the antibodies indicated. Film images are shown in (E) and pixel intensities from three independent experiments quantified in (F).