Cholesterol enhances ferroptosis resistance and unbalances myeloid/lymphoid lineage differentiation of HSCs. (A) Workflow of the RNA-seq analysis. (B) Ingenuity Pathway Analysis (IPA) showing the top changed molecular and cellular functions in LT-HSCs of HCD mice relative to CD mice. (C) Heat map showing fold change of cholesterol uptake, biosynthesis, esterification, and efflux genes in BM LT-HSCs of HCD mice. (D) GSEA of indicated gene sets in BM LT-HSCs of HCD mice. (E) IPA of cell death signaling pathways in BM LT-HSCs of HCD mice. (F–H) Flow cytometric analysis of membrane lipid peroxidation (F), 4-HNE and MDA contents (G), and ferrous ion deposition (H) in BM LT-HSCs of CD and HCD mice (n = 5). (I) Intracellular glutathione (GSH) and oxidized glutathione (GSSG) contents in BM LT-HSCs of CD and HCD mice (n = 3). (J) Ferroptosis resistance analysis of BM LT-HSCs from CD and HCD mice (n = 6). (K) Ferroptosis resistance analysis of LDLRlo and LDLRhi LT-HSCs (n = 6). (L) Ferroptosis resistance analysis of LT-HSCs with indicated treatment (n = 3). Data are mean ± SD. n.s., not significant. *p < 0.05, **p < 0.01. Two-tailed unpaired Student’s t-test unless stated otherwise. Two-tailed paired Student’s t-test (K). One-way ANOVA (L).