Cold stress‐induced ferroptosis involves the ASK1‐p38 pathway

A, B

Cells were pretreated with p38 inhibitors (10 μM SB203580, 10 μM SB202190), their inactive analog (10 μM SB202474), and a JNK inhibitor (1 μM SP600125) 30 min before cold stress application. Endogenous phospho‐ASK and phospho‐p38 signals were assessed by Western blot analysis using A549 lysates treated with cold stress on ice for 4 h.

C

Cells were pretreated with a JNK inhibitor (1 μM SP600125) 30 min before cold stress application. Cell death of A549 cells was determined by LDH assay after cold stress on ice for 8 h. Mean ± SEM, N = 5. Two‐way ANOVA followed by Bonferroni's multiple comparisons test was performed.

D

Lipid peroxidation levels in siRNA‐treated A549 cells were determined by Bodipy 581/591 after 4 h of cold stress on ice.

E

A549 cells were pretreated with p38 inhibitors (10 μM SB203580, 10 μM SB202190) for 30 min and then subjected to cold stress on ice for 4 h. Lipid peroxidation levels were determined by Bodipy 581/591.

F

Cold stress on ice was applied to A549 cells for 4 h, and total glutathione was measured. Mean ± SEM, N = 4. *P < 0.05 by unpaired two‐tailed Student's t‐test with Welch correction.

Figure EV1. Lipid peroxide‐ASK1‐p38 axis induces ferroptosis without mediating glutathione depletion.