Fig. 4: Both CoQ and squalene are required for cholesterol and desmosterol to suppress ferroptosis.

a Immunoblot analysis of indicated proteins in HT1080 cells after supplementation of cholesterol (40 µM) for indicated time. MβCD was used as vehicle control. b Quantification of mRNA levels of SQLE, FDFT1, HMGCR in HT1080 cells treated with cholesterol (40 µM) for 0 h, 2 h, 4 h or 6 h. c Immunoblot analysis of SQLE protein in HT1080 cells pre-treated with CHX (20 µg/ml) for 1 h, then co-treated with MβCD/cholesterol for indicated time. d Immunoblot analysis of SQLE protein in HT1080 cells with inhibitors pre-treated for 1 h followed by cholesterol (40 µM) for 4 h. Concentrations of inhibitors: MG132 (10 µM), CQ (10 µM), 3MA (5 mM), NH4Cl (5 mM). e Western blot analysis of knockout efficiency of SQLE protein in HT1080 cells. f Dose-dependent toxicity of RSL3 in HT1080 cells expressing sgRNA-Ctrl or sgRNA-SQLE with or without cholesterol (40 µM) treatment. g Western blot analysis of FDFT1 for HT1080 cells expressing siRNA-Ctrl or siRNA-FDFT1. h Dose-dependent toxicity of RSL3 in HT1080 cells expressing siRNA-Ctrl or siRNA-FDFT1. i Immunoblot assay of FDFT1 protein in wildtype and FDFT1 knockout HT1080 cells. j Cell death measurement of HT1080 cells expressing sgRNA-Ctrl or sgRNA-FDFT1 treated with RSL3 (500 nM) for 3 h. k Cell death measurement of HT1080 cells pre-incubated with TAK-475 (5 µM) for 12 h, then co-incubated with cholesterol (40 µM) for 3 h. RSL3 (500 nM) was added for 3 h after drug washout. l Cell death measurement of HT1080 cells expressing sgRNA-SQLE. Cells were pre-treated with TAK-475 (5 µM) for 12 h, then were incubated with RSL3 (500 nM) for 3 h. m Cell death measurement of HT1080 cells expressing sgRNA-Ctrl or sgRNA-FDFT1. Cells were pre-treated with 4-CBA (5 mM) for 12 h, then co-treated with cholesterol (40 µM) for 3 h. After drug washout, cells were incubated with RSL3 (500 nM) for 3 h. Uridine (200 µM) was continuously supplemented during the experiment. n Cell death measurement of HT1080 cells expressing sgRNA-FDFT1 or shRNA-COQ2. Cells were treated with cholesterol (40 µM) for 3 h, followed by washout, then treated with RSL3 (500 nM) for 3 h. Uridine (200 µM) was continuously supplemented during the experiment. o Western blot of FDFT1 and SQLE protein in HT1080 cells after transducing indicated expression plasmids. p Cell death measurement of HT1080 in (o). Cells were treated with cholesterol (40 µM) for 3 h. After washout, cells were incubated with RSL3 (500 nM) for 3.5 h. Data and error bars are mean ± SD, n = 3 (a–n) independent repeats. Significance in (a, c, d, h), was calculated using two-tailed unpaired Student’s t-test. Significance in (b, j–n) was calculated using a one-way ANOVA with Tukey’s post hoc test. Significance in (f, p) was calculated using a two-way ANOVA with Tukey’s post hoc test.