Fig. 1: ASS1 is essential for the p53 regulation of the cell cycle and genome integrity.
From: ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response
a, Quantification of the relative cell survival ability of HCT116 EV and ASS1-KO cells with and without Dox treatment. n = 8 independent experiments; EV, empty vector; ctrl, control. b, Quantification of the relative cell survival ability of normal fibroblasts (NF) and ASS1-deficient patient-derived skin fibroblasts (CTLN-I) with and without Dox. n = 4 independent experiments. c, Uracil:aspartate ratio of HCT116 EV and ASS1-KO cells with and without Dox. Treatment effect was significant (F1,8 = 14.7, P = 0.005) and similar in both cell lines (non-significant interaction between cell line and treatment; F1,8 < 0.01, P = 0.984). n = 3 biologically independent samples. d, Normalized total, pyrimidine and purine nucleotide levels as measured by LC–MS for HCT116 EV and ASS1-KO cells, with and without Dox (total: EV ctrl-EV Dox, P < 0.0001; ASS1-KO ctrl-ASS1-KO Dox, P = 0.0763. Pyrimidines: EV ctrl-EV Dox, P < 0.0001; ASS1-KO ctrl-ASS1-KO Dox, P = 0.0044. Purines: EV ctrl-EV Dox, P < 0.0001; ASS1-KO ctrl-ASS1-KO Dox, P = 0.1428). n = 3 biologically independent samples. e, Cell cycle of HCT116 EV and ASS1-KO cells, with and without Dox (G1: EV ctrl-EV Dox, P < 0.0001; EV ctrl-ASS1-KO ctrl, P = 0.0009; EV Dox-ASS1-KO ctrl, P < 0.0001; EV Dox-ASS1-KO Dox, P < 0.0001. G2M: EV ctrl-EV Dox, P < 0.0001; EV Dox-ASS1-KO ctrl, P < 0.0001; EV Dox-ASS1-KO Dox, P < 0.0001; ASS1-KO ctrl-ASS1-KO Dox, P < 0.0001). n = 3 biologically independent samples. f, Representative immunofluorescence images of γH2AX levels (Alexa Fluor 647) in control and Dox-treated HCT116 EV and ASS1-KO cells. Right panel: mean pixel intensity quantification (EV ctrl-EV Dox, P = 0.0049; EV ctrl-ASS1-KO ctrl, 0.0018; EV ctrl-ASS1-KO Dox, P < 0.0001; ASS1-KO ctrl-ASS1-KO Dox, P < 0.0001). n = 3 biologically independent samples. g, NF and ASS1-deficient patient-derived skin fibroblasts (CTLN-I) treated with and without Dox were immunoblotted for γH2AX, p53 and ASS1. Tubulin: loading control; MW, molecular weight. Right panel: Quantification of γH2AX and p53 protein expression levels comparing untreated NF and untreated CTLN-I cells; and for ASS1 protein expression levels in treated and untreated NF (NF, n = 2; CTLN-I, n = 3 biologically independent samples). h, Tail DNA percentage (%), in HCT116 EV and ASS1-KO cells with and without Dox treatment visualized using the comet assay. Representative fluorescent images for each sample are shown. Right panel: tail DNA percentage quantification. n = 600 biologically independent cells across 3 biologically independent samples (ctrl EV-ASS1-KO, P < 0.0001; Dox EV-ASS1-KO, P < 0.0001). Data are represented as the mean ± s.e.m. P values were determined by two-way ANOVA with Tukey’s honest significant differences method (in c and e), two-way ANOVA with Tukey’s multiple-comparison test (in d), ordinary one-way ANOVA with Sidak’s multiple-comparison test (in f and h) or unpaired two-tailed Student’s t-test (in a, b and g). Boxplot for h is min–max with the line at the median; shaded area, 50th percentile of each dataset.
