Fig. 3: ASNS modulates cell proliferation and senescence via asparagine.

a, b p53^+/+ and p53^−/− HCT116 cells expressing control or ASNS siRNA were injected subcutaneously into the dorsal flanks of twenty-eight nude mice. Tumour volumes (n = 7 mice in each group) were measured every 3 days after the tumours were visible (a). Representative images of xenografted tumours are shown (b). c HCT116 cells transfected with control or p53 siRNA were treated with increasing amounts of ASNase (0, 1 or 2 U per ml) for 48 h. Cell proliferation and protein expression were analysed. d, e p53^+/+ and p53^−/− HCT116 cells transfected with control or ASNS siRNA were cultured with or without 0.1 mM Asn for 3 days. Proliferation (d) and protein expression (e) were measured. f Proliferation of p53^+/+ and p53^−/− HCT116 cells treated with 0, 2 or 4 mM l-albizziine (l-Alb) or 0.1 mM Asn as indicated for 48 h. g Percentages of senescence-associated β-galactosidase (SA-β-gal)-positive p53^+/+ and p53^−/− HCT116 cells transfected with control or ASNS siRNA for 5 or 7 days. h HCT116 cells transfected with control or ASNS siRNA were cultured in medium with or without 0.1 mM Asn for 5 days. Percentages of SA-β-gal-positive cells are shown. i WI-38 cells transfected with control, ASNS siRNA and/or p53 siRNA were cultured for 3 days in the presence or absence of 0.1 mM Asn as indicated. Representative images, percentages of SA-β-gal-positive cells and protein expression are shown. j Protein expressions in WI-38 cells at different passages were measured. k The replicative lifespan of WI-38 cells treated with control, ASNS siRNA and/or 0.1 mM Asn as indicated. Arrow indicates the onset of senescence. Data are mean ± s.d., unpaired two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant. Source data are provided as a Source Data file.