Extended Data Figure 12. Neurons and cardiomyocytes acquire increased ether-phospholipid levels and elevated sensitivity to ferroptosis.

a. Scheme showing the experimental strategy for neuronal differentiation of SH-SY5Y cells, and representative images showing the cell morphology at indicated stages. RA, retinoic acid; BDNF, brain-derived neurotrophic factor; FBS, fetal bovine serum.

b. Immunoblot analysis showing the protein expression levels of relevant neuronal markers including MAP2 (microtubule associated protein 2), tyrosine hydroxylase, NeuN (neuronal nuclei antigen) and β-3-tubulin. Arrows indicate the band for the indicated full length protein, * indicates non-specific bands. β-Actin was used as a loading control. Representative results of experiment performed twice.

c. Viability curves of SH-SY5Y parental cells and cells at day 6 of neuronal differentiation under the treatment of indicated concentrations of RSL3 for 48 h. n=2 biologically independent samples. Representative results of experiment performed twice.

d. Fluorescent images showing lipid peroxidation levels reported by BODIPY-C11 oxidation. Representative images of experiment performed in duplicate.

e. Volcano plot showing free fatty acid lipidomics analysis in parental or differentiated SH-SY5Y cells. n=3 biological replicates for the parental condition, n= 4 biological replicates for the day 6 and day 12 differentiation condition. Two tailed Student’s T test. Multiple-testing adjustment was performed using the Benjamini-Hochberg method.

f. Immunofluorescence images showing the expression of cardiac troponin T, a marker of differentiated human cardiomyocytes, and NKX2.5, a cardiac-lineage specific marker in the cardiac progenitor (CP) cells and cardiomyocytes (CM). Scale bars indicate 15 µm. Representative results of experiment performed twice.

g. Viability curves of iPS cells and differentiated cardiomyocytes (CM) treated with indicated concentrations of ML210 or RSL3 for 24 h. n=4 biologically independent samples. Results from experiment performed once.

h. Bright field images showing cardiomyocytes treated with ML210 undergoing cell death. Scale bars indicate 30 µm. Representative results of experiment performed twice.

i. Bar plots showing relative viability of CMs treated with ML210 and indicated cell death inhibitors. z-VAD, z-VAD-FMK; Nec-1, necrostatin-1. n=3 biologically independent samples. Representative results of experiment performed twice. 0 µM vs 0.25 µM ML210 treated conditions with additional DMSO treatment only, p=0.00011. For 0.25 µM ML210 treated conditions, DMSO vs z-VAD, p=0.089; DMSO vs Nec-1, p=0.125; DMSO vs Fer-1, p=0.00061; DMSO vs Lip-1, p=0.00008. Two tailed Student’s t-Test.

j. Volcano plot showing free fatty acid lipidomics analysis in cardiac progenitors (CP) or differentiated cardiomyocytes (CM). n=2 biological replicates for CP, n=4 biological replicates for CM. Two tailed Student’s t-Test. Multiple-testing adjustment was performed using the Benjamini-Hochberg method.

k. qRT-PCR analysis showing the relative abundances of PEX3 (left) or AGPS (right) mRNAs in cardiomyocytes treated with the indicated siRNAs. n=2 biologically independent samples.

See Supplementary Information for uncropped immunoblot images. For viability curves and bar graphs, data center and error bars indicate mean±s.d..