Fig. 2: Ferroptosis inhibition abolishes spermine-induced cell death without altering the activation of RIPK3 and MLKL.

A, B Representative images of oxidized lipids (green) and non-oxidized lipids (red) with merged images and oxidation ratio in BMDMs treated with (A) spermine (Spm) for 9 h; (B) ras-selective lethal small molecule 3 (RSL3) for 6 h. C, D Real-time analysis and representative images of cell death in BMDMs treated with (C) Spm; (D) RSL3 for 6 h in the presence or absence of ferrostatin-1 (Fer-1). E Immunoblot analysis of released lactate dehydrogenase (LDH) and high mobility group box 1 (HMGB1) in cell culture media of BMDMs stimulated with RSL3 and Spm in the presence or absence of Fer-1. F, G Immunoblot analysis of phosphorylated receptor-interacting serine/threonine kinase 3 (pRIPK3), total RIPK3 (tRIPK3), phosphorylated mixed lineage domain-like pseudokinase (pMLKL), and total MLKL (tMLKL) in BMDMs treated with RSL3 and spermine in the presence or absence of (F) Fer-1; (G) necrostatin-1 (Nec-1). H Immunoblot analysis of pRIPK3, tRIPK3, pMLKL, and tMLKL in BMDMs after indicated times of RSL3 and Spm treatment. β-actin was used as an internal control (F–H). I Immunoblot analysis of released LDH and HMGB1 in the cell culture media of BMDMs after indicated times of RSL3 and Spm treatment. Data are shown as mean ± SEM (A–D). ****P < 0.0001 (one-way ANOVA; n = 4 from 4 biologically independent samples) (A and B). ****P < 0.0001 (two-tailed t test; n = 4 from 4 biologically independent samples) (C and D). Data are representative of at least three independent experiments (A–I). Scale bar, 100 μm (A–D). Source data are provided as a Source Data file.