Fig. 5: Role of cholesterol and bile acids in SF senescence.

a Functional annotation of all 279 genes included in the CellAge database. b Cholesterol-pathway-related genes identified by RNAseq of SF treated with 1 μM BMS for 7 days: red (up-regulated); green (down-regulated). Additional information on these genes is reported in Supplementary Table 3. c Protein–protein interaction (PPI) network constructed with STRING reveals the interactivity between senescence-related genes and the cholesterol pathway. d Levels of cholesterol and bile acids in SF treated with 1 μM BMS for 7 days were quantified by enzyme immune-assay. Data are mean ± SE (n = 11 for cholesterol, n = 11 for bile acids, Student’s t-test vs. control, Ctrl; *p < 0.05, **p < 0.01). e SF were treated with 1 μM BMS (B) with or without 1 μM atorvastatin (A) for 7 days and senescence determined by SA-βGal staining. Data are mean ± SE (n = 3, one-way ANOVA vs. control, Ctrl; *p < 0.05). f Gene expression of GPBAR1 and reference gene HPRT1 on differentiated human macrophages on two different donors is shown with expected bands at 63 and 130 bp, respectively. g Human macrophages were incubated with supernatants from senescent SF (SenS, from SF treated with 1 µM BMS for 7 days), with SenS supernatants plus 5β-cholanic acid (SenS + 5β), supernatants from control SF (CtrlS) or directly stimulated with 1 μM BMS for 5 days. Apoptosis was assessed by flow cytometry by measuring annexin A5 and propidium iodide staining. Data are mean ± SE (n = 3). h Activated caspase-3 was assessed by fluorescent microscopy on human macrophages treated as in panel g. Scale bars indicate 200 μm. Source data are provided as Source Data file.