Extended Data Fig. 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. R A, 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, Immunof luorescence images showing the expression of cardiac troponin T, a
marker of differentiated human cardiomyocytes, and NKX2.5, a cardiac-
lineage specific marker in the CP cells and CM. Scale bars,15 μm. Representative
results of experiment performed twice. g, Viability curves of iPS cells and
differentiated 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, 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 CP or differentiated 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 are mean ± s.d.