Cholesterol synthesis is predicted to be upregulated in long-term estrogen deprivation (LTED) cells. (a, b) Gene set enrichment analysis (GSEA) of (a) E2F activation signature and (b) cholesterol biosynthesis signature (Reactome Cholesterol Synthesis) in LTED variants. Differential expression (DE) genes used in GSEA were ranked by log2(fold change). c Growth of parental and LTED cells with treatment of 25-hydroxycholesterol (25-HC) and 27-hydroxycholesterol (27-HC). Parental cells were hormone-deprived before being seeded in hormone-deprived media (charcoal-stripped fetal bovine serum, or CSS). Cells were collected after 5-day treatment. Fold growth was compared with control group (data not shown), which was treated with vehicle (ethanol). Plots are representative of at least two independent experiments. Data are mean ± standard deviation (SD) of six replicates. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons was used to test the significance between 25-HC/27-HC–treated groups to the control groups (data not shown), *P <0.05, **P <0.01, ***P <0.001. d The activation z-score of sterol regulatory element-binding proteins (SREBPs) in LTED cells. The upstream regulator analysis was performed in individual LTED cell variants separately with Ingenuity Pathway Analysis (IPA) software. DE genes (absolute log2(fold change) > log2(1.5) and adjusted P value of less than 0.001) and their log2(fold change) were used as input. Upstream regulators with z-score of more than 2 were defined as “activated” (labeled in red). Abbreviation: ES enrichment score.