Functional validation of Cas9 expression and comprehensive lipid profiling in cyto-STARD7 cells. (a–c) Cyto-STARD7 HeLa cells were transduced with a Cas9-expressing lentivirus and selected with blasticidin to generate a stable, monoclonal population expressing Cas9. To test Cas9 activity, cells were transfected with a BFP-GFP reporter plasmid containing a guide RNA targeting GFP. Green and blue fluorescence were measured in untransfected HeLa cells (a), parental cyto-STARD7 cells lacking Cas9 (b), and cyto-STARD7 cells stably expressing Cas9 (c). In cells without Cas9, both green (GFP) and blue (BFP) signals are visible, indicating no gene editing. In contrast, in Cas9-expressing cells, ∼80% of cells lost green fluorescence but retained blue, demonstrating successful Cas9-mediated GFP KO. (d and e) Genes whose guide RNAs are depleted (d) or enriched (e) in chronic and acute ferroptosis screens in cyto-STARD7 HeLa cells. Lists include genes with FDR <0.05: 12 significant genes identified in the chronic screen (d) and 81 in the acute screen (e). Genes highlighted in bold red are part of the SCAP–SREBP and mevalonate pathways. (f–l) Levels of various lipid species, including lysophosphatidylcholine (lyso-PC), PC, lysophosphatidylethanolamine (lyso-PE), PE, PG, PI, diacylglycerol (DAG), sphingomyelin (SM), and several fatty acids (saturated: 16:0, 18:0; monounsaturated: 18:1; polyunsaturated: 20:4, 22:6) were measured in WT and cyto-STARD7 HeLa cells using mass spectrometry. Fatty acids were extracted from the polar phase using the methyl-tert-butyl ether (MTBE) method, while all other lipids were extracted from the lipid phase (n = 5 biological replicates). Mass spectrometry peak areas were normalized to the protein concentration of the corresponding cell pellets. Error bars represent the 95% confidence interval.