Extended Data Figure 7. AGPAT3 contributes to polyunsaturated ether phospholipid synthesis downstream of peroxisomes.

a. Immunoblot analysis of ACSL4 and LPCAT3 protein levels in 786-O cells expressing the indicated sgRNAs. Plot of experiment performed once.

b. Immunoblot analysis of ACSL4 and LPCAT3 protein levels in OVCAR-8 cells expressing the indicated sgRNAs. Plot of experiment performed once.

c. Immunoblotting of ACSL4, AGPS and FAR1 protein 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-sgRNA1^TetOn 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 AGPAT3-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 AGPAT3-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 AGPAT3-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 AGPAT3 mRNA expression in 786-O and 769-P cells expressing non-targeting negative control shRNA (shNC) or AGPAT3-targeting shRNAs. B2M was used as a loading control. n=3 biologically independent samples. Two-tailed student’s T-test; ns, not significant.

j. Viability curves for 786-O, and 769-P cells expressing shNC or AGPAT3-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 Agpat3^E176A cDNA construct.

l. Viability curves of AGPAT3-sg1-expressing OVCAR-8 cells rescued with sgRNA-resistant, wildtype (WT) mouse Agpat3 or Agpat3^E176A 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 center and error bars indicate mean±s.d..