Fig. 3: ACSS2 deficiency increases purine biosynthesis in senescence.

a ER:RAS-expressing cells were induced to senesce by adding 100 nM 4-OHT. At day 8 post-induction, cells were infected with lentivirus encoding non-targeting shRNA, ACSS2, or ACLY-targeted shRNAs. The puromycin-selected cells were harvested to perform targeted metabolomics analysis. Hierarchical clustering of significantly altered metabolites among groups was shown (n = 3 each group). b Top-ranked metabolic pathways in senescent cells with ACSS2 knockdown compared to control shRNA knockdown. P values were calculated using hypergeometric distribution (MetaboAnalyst v6.0). c, d Heat map analysis of the metabolites involved in the pyrimidine pathway (c) and the purine pathway (d). e ER:RAS-expressing cells were induced to senesce by adding 100 nM 4-OHT. On day 8 post-induction, cells were infected with lentivirus-encoding non-targeting shRNA or ACSS2-targeted shRNA. The puromycin-selected cells were harvested to measure cellular dNTP levels. f–h ER:RAS-expressing cells were induced to senesce by adding 100 nM 4-OHT. At day 8 post-induction, cells were supplemented with or without nucleosides or purine-only nucleosides for 14 days. Cells were stained for γH2AX (f). Three randomly imaged fields were analysed (g). The SASP mRNA expression was examined by qRT-PCR (h). Scale bar = 20 μm. Data represent mean ± s.d. of three biologically independent experiments. The P values were calculated using an unpaired two-tailed Student’s t-test (e) or One-way ANOVA (Tukey’s multiple-comparison test) (g, h). Pro, proliferating cells; Sen, RAS induced senescence; NS, nucleosides; A + G, Adenosine+Guanosine. Source data are provided as a Source Data file.