Fig. 1: Cytosolic Ca²⁺ increases and cell death during ferroptosis in a treatment-dependent manner.

Confocal images of NIH-3T3 cells treated with Erastin-1 (A) or RSL3 (B) in the presence or not of different cell death inhibitors. Pictures are representative of at least three independent experiments. Scale bar, 50 µm. Increase of Fluo-4 AM and PI-positive cells after 24 h of treatment with Erastin-1 (C, E) or RSL3 (D, F). Time course of the increase in the percentage of Fluo-4 AM and PI-positive cells upon treatment with Erastin-1 (G) or RSL3 (H), in the presence or not of Fer-1. The values represent the mean and the standard deviation of at least three independent flow cytometry experiments. An increase in cytosolic Ca²⁺ signal was detected using the Ca²⁺ indicator Fluo-4 AM, and plasma membrane breakdown was detected with PI. Fer-1 ferroptosis inhibitor ferrostatin-1, Nec-1s necroptosis inhibitor necrostatin-1s, zVAD pan-caspase inhibitor for apoptosis and pyroptosis. Concentrations: Erastin-1 (10 µM), RSL3 (2 µM), Fer-1 (2 µM), Nec-1s (10 µM), zVAD (20 µM).