Fig. 6: Changes in asparagine-aspartate homeostasis alter AMPK signalling.

a, b HEK293 cells (a) and HCT116 cells (b) were transfected with increasing amounts of ASNS siRNA for 3 days. Cellular Asn and Asp were determined and quantified by LC-MS. The ratios of Asn/Asp are shown (top left panel). Protein expression was determined (left panel), and the ratios of phosphorylated AMPK, total AMPK, phosphorylated p53 and total p53 are shown (bottom right panel). c, d HEK293 cells (c) and HCT116 cells (d) were transfected with increasing amounts of Flag-ASNS plasmids as indicated for 3 days. Cellular Asn and Asp were determined and quantified by LC-MS (top right panel). The ratios of Asn/Asp (top left panel) and protein expression (left panel), and the ratios of phosphorylated AMPK, total AMPK, phosphorylated p53 and total p53 are shown (bottom right panel). e, f C57BL/6 J mice xenografted s.c. with 5 × 10⁵ EL4-Luc-GFP cells were administered vehicle, Asn or Asp by either oral absorption (from drinking water, D.W.) or intraperitoneal injection (i.p.) of 500 μl every two days as indicated. The whole-mouse bioluminescence analysis was performed at 18 days post-injection. e Representative images of the mice are shown. The colour scale represents the intensity of the emitted luminescence. Each group includes 5 mice. Tumour volumes were measured by a calliper at 10, 14 and 18 days post-xenograft. Each group includes 5 mice and 10 tumours. f Expression levels of AMPK, p-AMPK, p53, p-p53 and p21 in xenografted tumours were analysed by western blotting. Data are mean ± s.d., unpaired two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant. Source data are provided as a Source Data file.