Panel a shows a vertical bar graph comparing cholesterol esters and cholesterol levels between wild-type and cyto-STARD7 groups. The x-axis represents different cholesterol esters and cholesterol, while the y-axis represents the area per nanogram of protein. Panel b shows a vertical bar graph comparing coenzyme Q10 levels between wild-type and cyto-STARD7 groups, with the x-axis representing coenzyme Q10 and the y-axis representing the area per nanogram of protein. Panel c shows a vertical bar graph comparing cholesterol and coenzyme Q10 levels under different conditions of Fato and fetal bovine serum treatments, with the x-axis representing these conditions and the y-axis representing the area per nanogram of protein. Panel d shows a vertical bar graph comparing coenzyme Q10 levels under different conditions of Fato treatment, with the x-axis representing these conditions and the y-axis representing the area per nanogram of protein. Panel e shows a vertical bar graph comparing coenzyme Q10 levels under different treatments over 24 and 72 hours, with the x-axis representing the treatment duration and the y-axis representing the area per nanogram of protein. Panel f shows a vertical bar graph comparing coenzyme Q10 enrichment under different treatments and time points, with the x-axis representing the treatment and time points and the y-axis representing the enrichment. Panel g shows a line graph depicting the dead cell area per phase area under different concentrations of ML210, with the x-axis representing ML210 concentration and the y-axis representing the dead cell area per phase area. Panel h shows a vertical bar graph depicting the dead cell area per phase area in HCT116 cells under different treatments, with the x-axis representing the treatments and the y-axis representing the dead cell area per phase area. Panel i shows a vertical bar graph depicting the dead cell area per phase area in SKMEL28 cells under different treatments, with the x-axis representing the treatments and the y-axis representing the dead cell area per phase area. Panel j shows a vertical bar graph depicting the dead cell area per phase area in HeLa cells under different treatments, with the x-axis representing the treatments and the y-axis representing the dead cell area per phase area. Panel k shows a line graph depicting the dead cell area per phase area under different concentrations of RSL3 and Mevastatin, with the x-axis representing RSL3 concentration and the y-axis representing the dead cell area per phase area. Panel l shows a vertical bar graph comparing relative cholesterol intensity between parental and single-guide RNA SCAP groups, with the x-axis representing the groups and the y-axis representing the relative cholesterol intensity. Panel m shows a line graph depicting the dead cell area per phase area over time under different treatments, with the x-axis representing time and the y-axis representing the dead cell area per phase area.
Kinetic assessment of CoQ and cholesterol upon SCAP activation and inhibition. (a and b) Cholesterol (a) and CoQ (b) levels measured via mass spectrometry from the lipid phase of WT and cyto-STARD7 HeLa cells (n = 5). The peak area is normalized to protein concentration. (c and d) HeLa cells were cultured with or without the SCAP inhibitor fatostatin (n = 4) at the indicated concentrations. As a positive control for cholesterol measurement, cells cultured in the absence of FBS were used to observe a decline in cholesterol levels (c). The peak areas of cholesterol (c) and CoQ (d) were normalized to protein concentration. (e) CoQ levels were measured using mass spectrometry in HeLa cells treated with either DMSO (orange) or the SCAP inhibitor fatostatin (blue) for 24 or 72 h (n = 3). The peak area for CoQ was normalized to the protein concentration in the samples. (f) HeLa cells were treated with either fatostatin or cholesterol (CR) at the indicated concentrations for 24 and 48 h (n = 5). After treatment for 24 h, the culture medium was replaced with DMEM containing 13C6-labeled glucose. Cells were then snap-frozen either 5 or 24 h after the 13C6-glucose incubation. To assess newly synthesized CoQ, lipids were extracted from the lipid phase, and 13C6 enrichment in CoQ molecules was measured using mass spectrometry. (g–j) Dose–response analysis of ferroptosis sensitivity in HCT116 colorectal cancer cells (h), SK-MEL-28 melanoma cells (i), and HeLa cells (WT and cyto-STARD7) (g and j). HeLa cells were treated with the SCAP inhibitor fatostatin at the indicated concentrations (0–5 µM) followed by ferroptosis induction using increasing concentrations of the GPX4 inhibitors RSL3 (100 nM, j) or ML210 (indicated concentration, g). (k) Dose-dependent sensitization of HeLa cells to RSL3-induced ferroptosis following treatment with the HMGCR inhibitor mevastatin (1 or 2 µM). In all experiments (g–k, and m), ferroptosis was suppressed by the lipophilic antioxidants ferrostatin-1 (Ferr, 1 µM) or liproxstatin-1 (Lip-1, 1 µM). (l and m) Generation and validation of SCAP KO HeLa cells. WT HeLa cells were transduced with lentiviral sgRNA against SCAP and selected with puromycin. (l) Quantification of total cholesterol levels via mass spectrometry in parental and SCAP KO polyclonal populations following 48 h of serum deprivation. (m) Real-time monitoring of cell death in SCAP KO cells using the Incucyte system. Cell death was quantified by normalizing the Sytox Green-positive area to the total phase-contrast area (n = 4). Error bars in a–m represent the 95% confidence interval (CI). P values are indicated above the respective data points. GPX4, glutathione peroxidase 4