Ferroptosis inhibitors prevent PCD induced by 55°C HS in Arabidopsis root hairs. (a) 6-d-old seedlings were preincubated with 1 µM Fer-1 (white bars), 10 µM CPX (gray bars), or DMSO (black bars). Cell death was induced by treating roots at 55°C or 77°C for 10 min, with H₂O₂ for 6 h, or with NaCl for 16 h. (b) 6-d-old seedlings were preincubated with CaCl₂ for 16 h, with 1 mM EGTA for 2 h, or with EGTA for 2 h and then with CaCl₂ for 16 h before inducing cell death by treating roots at 55°C for 10 min. (a and b) Root hairs were stained with Sytox green, and Sytox-positive cells (interpreted as dead cells) and Sytox-negative cells were quantified. Results are expressed as a percentage of dead cells. Data are the mean + SEM of three independent experiments. Bars with different letters denote statistical difference (one-way analysis of variance, P < 0.05). Also see Fig. S1. (c) 6-d-old seedlings were preincubated with Fer-1 analogues SR9-01 and SRS8-24 before treatment at 55°C. Root hairs were stained with Sytox green, and the number of Sytox-positive cells (interpreted as dead cells) and Sytox-negative cells was quantified to obtain the EC₅₀ of those compounds.