The bar graphs show the area of various lipid species in mitochondrial and non-mitochondrial fractions of cells treated with DMSO or fatostatin. Panel a to c show the mitochondrial fraction collected from an 8000 g pellet, with graphs for CoQ, lysoPE, PE, lysoPC, and PC. Panel d to i show the non-mitochondrial fraction collected from 12000 and 4000 g pellets, with similar graphs for CoQ, lysoPE, PE, lysoPC, and PC. Panel j to n shows the percentage of mitochondrial and extra-mitochondrial CoQ in parental, sgRNA SCAP, and DMSO-treated cells. Panel o and p show the relative CoQ10 intensity in cells treated with DMSO or mevastatin. Panel q shows the dead cell area after treatment with RSL3 and iFSP1. The heatmap in panel l shows the distribution of various lipid species in parental and sgRNA SCAP cells across different pellets. Each bar graph has the area on the y-axis and the lipid species on the x-axis. The heatmap uses a color scale to represent the relative abundance of lipid species.
CoQ distribution upon mevalonate pathway inhibition. (a–i) HeLa cells (30 × 106) were treated with DMSO or the SCAP inhibitor fatostatin (5 µM) for 24 h and used for the enrichment of mitochondria through differential centrifugation (n = 5). First, cells were centrifuged at 8,000 × g to pellet mitochondria, which were then sonicated in methyl-tert-butyl ether (MTBE) buffer (a–c). The supernatant was further centrifuged at 12,000 × g to isolate heavy membranes, followed by a 20,000 × g centrifugation to isolate non-mitochondrial membranes (d–i). Both pellets were resuspended in MTBE and sonicated for lipid extraction. The lipid phase was separated from the polar phase and analyzed for CoQ (a, d, and g), lysoPE and PE (b, e, and h), and lysoPC and PC (c, f, and i). (j and k) The percentage of mitochondrial CoQ (j) and extramitochondrial CoQ (k) was calculated in cells treated with either DMSO or fatostatin for 24 h. Total CoQ levels in cells are shown as the sum of peak areas from the 8,000 × g, 12,000 × g, and 20,000 × g pellets in each replicate. To calculate mitochondrial CoQ (j), the peak area from the 8,000 × g pellet was divided by total CoQ, and for extra-mitochondrial CoQ (k), the combined peak areas from the 12,000 × g and 20,000 × g pellets were divided by total CoQ. (l–p) Lipidomic profiling and CoQ quantification in parental and SCAP KO polyclonal HeLa cells. (l) Heatmap illustrating the subcellular distribution of CL species, TG, PC, and PI across the different fractions. The selective enrichment of CL in the 8k pellet validates the successful isolation of mitochondria. Gray cells in the heatmap represent lipids that were below the limit of detection. (m and n) CoQ levels in parental vs. SCAP−/− cells and o and p, in WT HeLa cells treated with DMSO or the HMGCR inhibitor mevastatin for 48 h. (q) Epistasis analysis of mevalonate pathway inhibition and FSP1 activity. Real-time cell death monitoring in HeLa cells treated with mevastatin at indicated concentration (alone) or in combination with the FSP1 inhibitor iFSP1 (5 µM) following ferroptosis induction with RSL3. Cell death was quantified by normalizing the Sytox Green-positive area to the total phase-contrast area. Error bars in a–p represent the 95% confidence interval (CI). P values are indicated above the respective data points.