Fig. 5: ACSS2 promotes acetylation of PAICS and decreases its protein stability.

a Proliferating and OIS cells with or without ACSS2 knockdown were harvested. Co-immunoprecipitation analysis of endogenous PAICS and ACSS2 in extracts from the indicated IMR90 cells was performed and subjected to immunoblots of the indicated proteins. b Purified human protein of ACSS2 and PAICS was subjected to in vitro pull-down assay. Immunoblot of the indicated proteins was shown. c Proliferating and OIS cells were infected with HA-PAICS encoding lentivirus, and then treated with or without ACSS2i for 48 h. PAICS protein was immunoprecipitated, and the acetylation was analysed by immunoblot with the indicated antibody. d HEK293T cells were transfected with HA-tagged wildtype (WT) or K36R, K47R, and K36RK47R mutants of PAICS. HA-PAICS protein was immunoprecipitated, and the acetylation was analyzed by immunoblot with the indicated antibody. e, f ER:RAS-expressing cells were induced to senesce by adding 100 nM 4-OHT. On day 8 after the induction, the senescent cell was infected with lentivirus encoding non-targeting shRNA or ACSS2 targeted shRNA (e) or treated with ACSS2i for 48 h (f). The cells were harvested and analysed by immunoblot for expression of the indicated proteins. g, h Proliferating and senescent cells described in (e) were treated with CHX (100 μg/mL) for 0, 6, or 12 h, and the expression of the indicated proteins was analysed by immunoblot (g) and quantified (h). i OIS cells were subjected to co-immunoprecipitation analysis of endogenous PAICS and PCAF, immunoblots of the indicated proteins were shown. j, k OIS cells expressing non-targeting shRNA or PCAF-targeted shRNA were infected with HA-PAICS encoding lentivirus. PAICS protein was immunoprecipitated, and the acetylation was analyzed by immunoblot with the indicated antibody (j) and quantified (k). l Proliferating and OIS cells expressing non-targeting shRNA or PCAF-targeted shRNA were subjected to immunoblots of the indicated proteins. Data represent mean ± s.d. of three biologically independent experiments. The P values were calculated using an unpaired two-tailed Student’s t-test (k). Representative of n = 3 independent experiments were shown (a–g, i, j, l). Source data are provided as a Source Data file.