Extended Data Fig. 8: Polyamine biosynthesis is critical for tumour cell proliferation.
From: p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis
a, HCT116 cells treated with control siRNA, triple knockdown siRNA (CPS1, OTC and ARG1 siRNA; siTriple) or 200 μM DFMO were cultured in medium containing increasing amounts of putrescine (0, 100 and 500 μM) for 6 days. Cell proliferation was determined by cell counting. b, Colony formation assay of p53+/+ HCT116 cells treated with control siRNAs, triple knockdown siRNA or DFMO were cultured in medium containing increasing amounts of putrescine as indicated. Numbers of colonies were quantified (left). Data are mean ± s.d. Colonies were stained with crystal violet at day 12. Representative images of colonies are shown (right). c, p53+/+ and p53−/− HCT116 cells were cultured in medium containing DMSO or 0.25 mM DMFO for indicated times. Cell proliferation was determined by cell counting. d, Colony formation assay of p53+/+ and p53−/− HCT116 cells treated with DMSO (0 mM) or increasing amounts of DMFO (0.25 mM, 2 mM) as indicated. Representative images of colonies stained with crystal violet at day 14 are shown. e, Colony formation assay of p53+/+ and p53−/− HCT116 cells transfected with two different sets of ODC1 siRNAs (siODC #1 and #2) or control siRNA as indicated. Numbers of colonies with a diameter greater than 15 μm were quantified (left, bottom). ODC knockdown efficiency was determine by western blot analysis (left, top). Representative images of colonies stained with crystal violet at day 12 are shown (right). f, g, Colony formation assay of A549 cells (f) and H1299 cells (g) transfected with two different sets of ODC1 siRNAs or control siRNA as indicated. Numbers of colonies were quantified (left, top) and ODC knockdown efficiency was determined by western blot analysis (left, bottom). Representative images of colonies stained with crystal violet at day 14 are shown (right panel). h, Related to Fig. 3f. Expression of ODC and p53 in xenograft tumours derived from p53+/+ and p53−/− HCT116 transfected with control siRNA or ODC1 siRNA. Data are mean ± s.d. of one representative experiment with n = 3 (a, b), n = 6 (d, e) or n = 4 (f, g) cultures out of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons test (a, b), or one-way ANOVA followed by Dunnett’s multiple comparisons test (d–g). See Supplementary Fig. 1 for gel source data.
