Extended Data Fig. 7: p53 induces ammonia accumulation to suppress tumour cell proliferation and polyamine biosynthesis.
From: p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis
a, Ammonia levels in p53+/+ and p53−/− HCT116 cells, U2OS cells expressing p53 shRNA or control shRNA, or control CRISPR and p53 CRISPR HepG2 cells was quantified. b, p53+/+ and p53−/− HCT116 cells, or U2OS p53 shRNA and U2OS control shRNA cells were treated with 0, 5, or 10 mM NH4Cl as indicated for 24 h. The levels of cellular ammonia were assayed. c, Ammonia levels in liver derived from p53+/+ and p53−/− mice loaded with or without 0.25 M NH4Cl for 6 days (n = 5 mice per treatment, total n = 20). d, p53+/+ and p53−/− HCT116 cells, or U2OS cells transfected with p53 shRNA or control shRNA were treated with 0, 5 or 10 mM NH4Cl as indicated for 24 h. Proliferation inhibition was assayed. e, p53+/+ and p53−/− HCT116 cells treated with control siRNA, or with CPS1, OTC and ARG1 siRNAs as indicated were incubated with increasing amounts of NH4Cl (0, 5 or 10 mM) for 24 h. Proliferation inhibition was determined by cell counting. f, Colony formation assay of p53+/+ and p53−/− HCT116 cells treated with increasing amounts of NH4Cl (0, 5 or 10 mM) as indicated. Data are mean ± s.d. of one representative experiment (n = 6 cultures per treatment). Colonies were stained with crystal violet at day 14. Representative images of the colonies are given. g, Related to Fig. 3c. p53+/+ and p53−/− HCT116 cells were treated with increasing amounts of NH4Cl for 24 h. The levels of putrescine were determined and quantified by high-performance liquid chromatography (HPLC) and peak images for each of the polyamines are given. 1,8-Diaminooctane was used as an internal control. h, k, l, U2OS cells stably expressing control shRNA or p53 shRNA were treated with increasing amounts of NH4Cl as indicated for 24 h. The levels of putrecine (h), spermidine (k) and spermine (l) were determined and quantified by HPLC. i, j, Related to Fig. 3c. The levels of spermidine (i) and spermine (j) in p53+/+ and p53−/− HCT116 cells treated with increasing amounts of NH4Cl for 24 h were determined and quantified by HPLC. Data are mean ± s.d. of n = 3 (a, b, d, h–l) or n = 2 (e) technical replicates of one out of three independent experiments, or n = 3 biologically replicates (h–l). *P < 0.05, **P < 0.01, two-tailed Student’s t-test (a), two-way ANOVA (b–d, h–l) or one-way (f) ANOVA followed by multiple t-test using Tukey’s or Dunnett’s method. Soft agar experiments (f) were performed twice. Liver ammonia assay (c) was performed once. All other experiments were performed three times.
