Fig. 3: Nuclear ASS1 contributes to fumarate generation and interacts with SMARCC1.
From: ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response
a, Isotopic tracing of labeled M + 4 fumarate (AUC) generated from M + 4 aspartate (AUC) in the nuclei was performed on control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells. n = 2 biologically independent samples, ASS1-KO, n = 1. b, Cell survival of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells were incubated with (+Fum) or without Fumarate (1 mM) using Apotracker. n = 3 biologically independent samples (EV ctrl-Dox, P < 0.0001; ASS1-KO ctrl-Dox, P < 0.0001; ASS1-KO Dox-Dox+Fum, P = 0.0244). c, Pathway analysis of ASS1 protein interactors from the nuclear fraction of HCT116 WT cells, after immunoprecipitation–MS using ASS1 antibody of the nuclear fraction of HCT116 WT cells. Chromatin-remodeling pathways related to SWI/SNF are highlighted in yellow. n = 3 biologically independent samples. d, Nucleosomes from control and Dox-treated HCT116 WT cells isolated by MNase digestion and immunoblotted for ASS1 and p53. Markers: H3, nuclear; GADPH, cytoplasmic. n = 3 independent experiments. e, Control and Dox-treated HCT116 WT cells assayed for the total, soluble and chromatin-bound fraction and immunoblotted for ASS1 and p53. Markers: H3, nuclear; GADPH, cytoplasmic. n = 3 independent experiments. f, Interactions between ASS1 and SMARCC1 of control and Dox-treated HCT116 WT cells visualized using PLA. Right panel: quantification of total puncta and nuclear puncta. n = 100 biologically independent cells across three biologically independent samples. g, interactions between ASL and SMARCC1 of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells visualized using a PLA. Right panel: quantification of nuclear puncta. n = 200 biologically independent cells across three biologically independent samples. Data are represented as the mean ± s.e.m. ns, not significant. P values were determined by one-way ANOVA with Tukey’s multiple-comparison test (in g), two-way ANOVA on the log10 transformed values (in b) or unpaired two-tailed Student’s t-test (in a, c and f). Boxplot in f and g is min–max, with the line at the median and the shaded area representing the 50th percentile of each dataset.
