Extended Data Fig. 7 | AGPAT3 contributes to PUFA-ePL synthesis
downstream of peroxisomes. a, Immunoblot analysis of ACSL4 and LPCAT3
levels in 786-O cells expressing the indicated sgRNAs. Plot of experiment
performed once. b, Immunoblot analysis of ACSL4 and LPCAT3 levels in
OVCAR-8 cells expressing the indicated sgRNAs. Plot of experiment performed
once. c, Immunoblotting of ACSL4, AGPS and FAR1 levels in the indicated
OVCAR-8 cell lines. Representative result of experiment performed in
duplicate. d, Viability curves of OVCAR-8 cells expressing sgNC or sgRNAs
targeting the gene of interest (GOI) as indicated on top of each graph, and
transduced with doxycycline (dox)-inducible ACSL4 sgRNA1Te t O n construct.
These cells were pre-treated with vehicle or dox, and then treated with
indicated concentrations of ML210, or ML210+Fer-1 for 72 h. n = 3 biologically
independent samples. Representative results from experiment performed
twice. e, Volcano plot showing the changes in phospholipid levels in sgNC or
AG PAT3-targeting sgRNA expressing 769-P cells. n = 3 biologically independent
samples. Two tailed Student’s t-test. Multiple-testing adjustment was
performed using the Benjamini–Hochberg method. f, Viability curves for 769-P
cells expressing sgNC or AG PAT 3-targeting sgRNAs treated with indicated
concentrations of ML210 or RSL3 for 48 h. n = 4 biologically independent
samples. Representative results from experiment performed in triplicate.
g, Viability curves for 786-O and OVCAR-8 cells expressing sgNC or AG PAT 3-
targeting sgRNAs treated with indicated concentrations of RSL3 for 48 h. n = 3
(OVCAR-8) or n = 4 (786-O) biologically independent samples. Representative
results from experiment performed in triplicate. h, Fluorescent imaging
showing nuclear staining by Hoechst 33342 in OVCAR-8 cells with the indicated
genetic perturbations and treated with vehicle (DMSO) or indicated
concentrations of ML210 for 5 days. Representative images from experiment
performed once, and each condition has three biological replicates. i, qRT–PCR
analysis of relative AG PAT 3 mRNA expression in 786-O and 769-P cells
expressing non-targeting negative control shRNA (shNC) or AG PAT 3-targeting
shRNAs. B2M was used as a loading control. n = 3 biologically independent
samples. Two-tailed Student’s t-test. j, Viability curves for 786-O, and 769-P
cells expressing shNC or AG PAT 3-targeting shRNAs treated with indicated
concentrations of ML210 or RSL3 for 48 h. n = 4 biologically independent
samples. Representative results from experiment performed in triplicate.
k, Nucleotide traces in Sanger sequencing analysis showing the point mutation
introduced in the mouse Agpat3E 176A cDNA construct. l, Viability curves of
AG PAT 3 sg1-expressing OVCAR-8 cells rescued with sgRNA-resistant, wild-type
mouse Agpat3 or Agpat3E 176A mutant cDNA and being treated with indicated
concentrations of RSL3 for 24 h. n = 4 biologically independent samples.
Representative data of experiment performed twice. β-Actin or GAPDH was
used as a loading control. See Supplementary Information for uncropped
immunoblot images. For viability curves and bar graphs, data are mean ± s.d.