Fig. 2: Mitochondrial 15LO1 pathway activity drives compartmentalized ferroptosis.

A IL-13 induces ferroptosis-associated 15LO1 protein expression in the mitochondrial fractions. HAECs were stimulated with/without IL-13 for 7 days. Cytosolic and mitochondrial fractions were isolated by ultracentrifugation for WB analysis. Representative images from n = 3 biologic replicates, with data quantification analysis in Supplementary Fig. 2A–F. B IL-13 increases 15LO1/ATPsynthase colocalized puncta in HAECs in vitro. Top panel: IF/CF staining. Red: ATPsynthase; Green: 15LO1; Blue: DAPI; Yellow: colocalization. Bottom panel: co-localized 15LO1/ATPsynthase puncta in yellow. Lower left corner digitally magnified. Representative images from n = 3 biologic replicates, with quantitative analysis in Supplementary Fig. 2H. C 15LO1/ATPsynthase colocalization (IF/CF) in freshly blushed HAECs from healthy and asthmatic participants. Green: ATPsynthase; Red: 15LO1; Blue: DAPI; Yellow: colocalization. White arrows: cilia area. Representative images from n = 3 biologic samples from individual donors in each group. D IL-13 increased oxPE in mitochondrial fractions in HAECs in vitro as normalized to the total intracellular phospholipid (PL) or (E) cardiolipin (CL) by LC/MS analysis. Significance analyzed by paired T-testing of n = 6 biological replicates. F The ratio of 15-HpETE-PE to 15 HETE-PE is higher in freshly brushed HAECs from severe asthmatic as compared to healthy control participants (one way ANOVA) (G) and higher in those participants (mild to severe asthma) with high 15LO1 protein (by WB). Data are presented as mean ± s.d, and differences determined by one-way ANOVA (F) and unpaired t-test (G) based on n = 3–5 biological samples from individual donors. Source data are provided as a Source Data file.