Fig. 3: Cholesterol and desmosterol promote ferroptosis resistance partly through FSP1-CoQ10 axis.

a, b Mass spectrometric analysis of CoQ10 levels of HT1080 cells treated with cholesterol (40 µM), desmosterol (40 µM) for 6 h, or 4-CBA (5 mM) for 12 h. c, d Cell death measurement of HT1080 cells incubated with RSL3 with or without 4-CBA and cholesterol or desmosterol. Cells were pre-incubated with 4-CBA (5 mM) for 12 h, then co-incubated with cholesterol (40 µM) (c) or desmosterol (40 µM) (d) for 3 h, after which drugs were washed out and continued to be co-incubated with 4-CBA (5 mM) and RSL3 (500 nM) for 4 h. Uridine (200 µM) was continuously supplemented during the experiment. e, f Cell death measurement of wildtype and shCOQ2 HT1080 cells in the presence of RLS3. Cells were pre-treated with cholesterol (40 µM) (e) or desmosterol (40 µM) (f) for 3 h, followed by thorough washout, and then treated with RSL3 (500 nM) for 4 h. Uridine (200 µM) was continuously supplemented during the experiment. g Cell death measurement of HT1080 cells pre-incubated with BQR (50 µM) for 12 h, then co-incubated with cholesterol (40 µM) for 3 h. RSL3 (500 nM) was added for 4 h after drug washout. h western blotting for FSP1 protein in indicated HT1080 cells. i, j Cell death measurement of wildtype or FSP1 knockout HT1080 cells. Cells were pre-treated with cholesterol (40 µM) (i) or desmosterol (40 µM) (j) for 3 h, then treated with RSL3 (500 nM) for 4 h after drug washout. k Cell death measurement of HT1080 cells pre-incubated with CoQ10 (250 nM) for 12 h, then co-incubated with RSL3 (500 nM) for 4 h. l–o Quantification of mRNA levels of PDSS1 and COQ2 in HT1080 cells treated with cholesterol (40 µM) (l, m) or desmosterol (40 µM) (n, o) for 0 h, 2 h, 4 h or 6 h. Data and error bars are mean ± SD, n = 3 (a–o) independent repeats. Significance in (k) was calculated using two-tailed unpaired Student’s t-test. Significance in (f, g, i–j, l–o) was calculated using a one-way ANOVA with Tukey’s post hoc test.