Fig. 6: PAICS interacts with LC3 and undergoes lysosomal degradation during cellular senescence. | Nature Communications

Fig. 6: PAICS interacts with LC3 and undergoes lysosomal degradation during cellular senescence.

From: ACSS2 drives senescence-associated secretory phenotype by limiting purine biosynthesis through PAICS acetylation

Fig. 6

a, b ER:RAS-expressing cells were induced to senesce by adding 100 nM 4-OHT. At day 8, senescent cells were treated with or without MG132 (0.1 μM) (a) or BafA1 (30 nM) for 48 h (b); the cells were harvested and analysed for expression of the indicated proteins by immunoblot. c OIS cells expressing non-targeting shRNA or ATG7 targeted shRNA were harvested for expression of the indicated proteins by immunoblot. d–f Co-immunoprecipitation analysis of endogenous PAICS and LC3 was performed in proliferating and OIS cells (d), and the PAICS or LC3 IP bands were normalized to the LC3 or PAICS IP and the input bands, respectively (e, f). g–i HA-tagged WT or mutant PAICS and FLAG-tagged LC3 were co-transfected into HEK293T cells and analysed by co-immunoprecipitation (g), the HA or FLAG IP bands were normalized to the FLAG or HA IP and the input bands respectively (h, i). j–l HA-tagged WT PAICS and FLAG-tagged WT or mutant LC3 were co-transfected into HEK293T cells, and analysed by co-immunoprecipitation (j), the HA or FLAG IP bands were normalized to the FLAG or HA IP and the input bands respectively (k, l). m Schematic of the three LIR motifs in PAICS protein. n–p HA-tagged WT or LIR-motif-mutant PAICS and FLAG-tagged LC3 were co-transfected into HEK293T and analysed by co-immunoprecipitation (n), the HA or FLAG IP bands were normalized to the FLAG or HA IP and the input bands respectively (o, p). q, r HA-tagged WT or LIR-motif-mutant PAICS was transfected into HEK293T. At 48 h post-transfection, cells were treated with CHX (100 μg/mL) for 0, 6, or 12 h, and the expression of the indicated proteins was analysed by immunoblot (q) and quantified (r). 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, f, h, i) or One-way ANOVA (Tukey’s multiple-comparison test) (k, l, o, p). Representative of n = 3 independent experiments were shown (a–d, g, j, n, q). Source data are provided as a Source Data file.

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