Extended Data Fig. 3|. Distal CB pathway regulates ferroptosis independent of cholesterol and known ferroptosis defense system.

a, Levels of cholesterol in WT and CYP51A1, MSMO1, EBP, SC5D, DHCR7 KO HEK293T cells. b, Relative concentration of cholesterol in FBS and LPDS measured by Amplex^® Red Cholesterol Assay Kit. c, The concentrations of cholesterol in FBS and LPDS detected by LC-MS/MS. d, e, Cell viability in HEK293T (d) and HT1080 (e) cells treated with RSL3 for 6–8 h after pretreatment with or without cholesterol (25 μM) for 24 h in FBS- or LPDS-supplemented medium. n=2 biological replicates. f, g, The protein levels of GPX4, FSP1, DHODH, ACSL4 and SLC7A11 in WT SC5D KO (f) and DHCR7 KO (g) HEK293T cells detected by immunoblotting. h, i, GSH/GSSG level measurement in WT, SC5D KO (c) and DHCR7 KO (d) HEK293T cells. j, k, FACS analysis of Fe²⁺ using FerroOrange probe by measuring MFI values in WT, SC5D KO (j) and DHCR7 KO (k) HEK293T cells. l, m, Levels of squalene (l) and CoQ₁₀ (m) in WT, SC5D KO and DHCR7 KO HEK293T cells. Data are representative of two (a-c, l, m) and three (d-k) independent experiments. Data are mean ± s.d. of n=3 biological replicates (a-c, h-m). Statistical analysis was performed using one-way ANOVA (a, h-m) or unpaired two-tailed t-tests (b and c); ****P < 0.0001; ns, not significant.