Extended Data Fig. 2: p53 transcriptionally regulates urea cycle gene expression.
From: p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis
a, Gene expression in U2OS cells expressing p53 shRNA or control shRNA was analysed by qRT–PCR and normalized to ACTB. b, HEK293 cells were transfected with vector control plasmid (0 μg) or increasing amount of Flag-tagged p53 plasmids for 24 h. The expression of CPS1, OTC, ARG1, p53, p21 and ACTB was determined by qRT–PCR. c, The abundance of CPS1, OTC, ARG1, p53 and p21 transcripts in p53−/− HCT116 cells that were transfected with the p53(A138V) mutant and cultured at 32 °C or 37 °C was determined by qRT–PCR analysis (bottom). The expression of p53 and p21 was further analysed by western blot (top). d, Western blot analysis of CPS1, OTC or ARG1 expression in p53-depleted and control HCT116 or U2OS cells as indicated. e, Protein expression of CPS1, OTC, ARG1, p53 and p21 in human hepatoblastoma HuH-6 cells control CRISPR and p53 CRISPR cells was determined by western blot analysis. f, Western blot analysis of CPS1, OTC, ARG1 and AKT expression in different tissues derived from either p53+/+ or p53−/− mice. g, Enzyme activities of CPS1, OTC and ARG1 in U2OS cells expressing p53 shRNA or control shRNA were determined (see Supplementary Methods). Data are mean ± s.d. of one representative experiment, with n = 5 or n = 4 technical replicates. Experiments were repeated three times with similar results. h, The genomic structure of human CPS1, OTC and ARG1. Shown are the exon and intron organization, and the consensus p53 response element. i, j, Luciferase reporter assay with indicated response element constructs in human HEK293T cells co-transfected with or without the p53 expression vector. Data are mean ± s.d., n = 3. Consistent with previous findings, p53 is shown to suppress the luciferase expression driven by the genomic regions of ME1 and PDK2, and promote luciferase expression driven by the EPCAM genomic region. Renilla vector pRL-CMV was used as a transfection internal control. Relative luciferase activity (fold change) is shown. k, qRT–PCR analysis of mRNA expression of CPS1, OTC and ARG1 in HCT116 cells left untreated (0 μM) or treated with increasing amounts of the p53 inhibitor pifithrin-α (PFT-α) for 24 h (top). The expression of p53 and p21 was analysed by western blot (bottom). l, qRT–PCR analysis of mRNA expression of CPS1, OTC, ARG1, p53 and p21 in HepG2 cells left untreated (0 μM) or treated with increasing amounts of Nutlin-3 for 24 h (top). The expression of p53 and p21 was analysed by western blot (bottom). Data are mean ± s.d. of three biologically independent samples per treatment (a), or n = 3 technical replicates of one out of three independent experiments (b, c, i–l). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test (a–c, g, i, j) or two-way ANOVA followed by Tukey’s multiple comparisons test (k, l). See Supplementary Fig. 1 for gel source data.
