Extended Data Fig. 3: ASL participates in the nuclear generation of fumarate.

a, Livers from control and ASL-KO (ASL^neo/neo) were fractionated and immunoblotted for ASS1. H3 and GAPDH are used as a nuclear and cytoplasmic marker, respectively. (n = 4 biologically independent samples) b, Control and dox-treated HCT116 WT cells were stained for ASL using Alexa Fluor 594 (red). Nuclei were counter-stained with DAPI (blue). Scale bar, 10 μm. (n = 3 independent experiments) c, Control and dox-treated HCT116 WT cells were fractionated and immunoblotted for ASL and p53. Markers: Vinculin - cytoplasmic; H3 nuclear (n = 3 biologically independent samples) d, 1,100 bp surrounding the ASL transcription start site (GRCh38 chr7:66,075,357-66,076,456) taken from the UCSC genome browser (http://genome.ucsc.edu)⁶ The tracks shown: ReMap Altas of Regulatory regions filtered for TP53, TP63, TP73 binding sites (only TP53 are found)⁶²; MatInspector (Genomatix Genome Analyzer⁶³)predicted P53 binding sites; and RefSeq mRNAs. A binding site in HCT116 can be found in the proximal promoter, which overlaps a predicted binding site as well as binding peaks found in other cell lines. e, Left panel: Interactions between ASS1 and ASL were visualised in control and dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells using a proximity ligation assay. Scale bar, 10 μm. (P-values: Total: <0.0001, Nuclear: 0.0446) Right panel: Quantification of total puncta and nuclear puncta. (n = 100 biologically independent cells). f, Fractional labelling of fumarate M + 4/all masses generated from ¹³C₄-aspartate in purified nuclei isolated from mice’s livers with and without ASS1. (Ass1^Flox/Flox ; Ass1^Flox/Flox AlbCre: n = 1 in each group. biologically independent mouse) g, Schematic depiction of the potential sources of fumarate M + 4 from labelled ¹³C-aspartate M + 4. h, Control and dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells were incubated with (+ Fum) or without Fumarate (1 mM), and survival was measured and quantified using XTT. (n = 6 biologically independent experiments) (P-value: EV Ctrl-EV Dox:0.0195; EV Dox-EV Dox+Fum:0.4432; ASS1-KO Ctrl-ASS1-Dox:<0.0001; ASS1-KO Dox-ASS1-KO Dox+Fum:<0.0001) Data are represented as the mean ± s.e.m. ns, not significant. P values were determined by RM one-way ANOVA (h), unpaired two-tailed Student’s t-test (e), or Tukey’s multiple-comparison test (f). Box plot for e is min-max with the line at the median and the shaded area represents 50 percentile of each dataset.