Fig. 3: SLC13A3 regulates intracellular glutathione and leucine in β-catenin-activated liver cancer cells.

a Cellular accumulation of ¹³C₂,¹⁵N-glutathione (GSH) in HEK293 cells. HEK293-SLC13A3 and HEK-EV cells were incubated with isotope-labeled GSH at concentrations from 10 nM to 10 mM for 20 min. Saturable GSH uptake was calculated using the difference of GSH accumulation in HEK293-SLC13A3 and HEK-EV cells. The data were fit to a Michaelis–Menten equation. Data are presented as the mean ± SEM for a representative experiment (n = 3 independent experiments). b Cellular uptake of ¹³C₂,¹⁵N-glutathione (GSH) (5 and 10 μM) was significantly higher in HEK-SLC13A3 cells (red) compared to HEK293-EV cells (gray). Cells were incubated in the uptake buffer containing GSH for 20 min. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. c Representative western blots of SLC13A3, SLC7A5, and SLC3A2 in HepG2 and SNU398 cell. Stably control (shNC) and SLC13A3-knockdown (sh#1) cells were established by infection with shRNA lentivirus. Blots were representative of three independent experiments (two replicates per group for each experiment). d Cellular uptake of 2 mM leucine in HepG2 and SNU398 cells. Stability control (shNC, gray) and SLC13A3-knockdown cells (sh#1, blue) were incubated with 2 mM leucine for 20 min. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. e qRT-PCR and western blot analyses of MYC expression in HepG2 and SNU398 cells. Stably control (shNC, gray) and SLC13A3-knockdown (sh#1, blue and sh#2, dark blue) cells were established using respective shRNA lentivirus. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. Blots were representative of three independent experiments. f Methyl-DNA immunoprecipitation (MeDIP)-PCR detection of the MYC promoter methylation. DNA was immunoprecipitated with 5-methylcytosine antibody (blue) or IgG (gray), and purified according to the manufacturer’s protocol. Input DNA prior to immunoprecipitation was used as the positive control. Precipitated and input DNAs were amplified using primers for the CpG islands in the MYC promoter region, and PCR products were visualized by gel electrophoresis. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. g Mouse study design. Mice were given a leucine-deficient diet (Leu-) or leucine-supplemented drinking water (1.5% leucine in drinking water, Leu+). Liver tumor was induced by hydrodynamic tail vein injection (HTVi) of c-Met/β-catenin plasmids or AKT/β-catenin plasmids. h Mice were euthanized when they developed a high burden of liver tumors. The log-rank test was used to compare overall survival between groups (Gray, control; Blue, Leu+; Orange, Leu-). ns, non-statistically significant. i, j Representative gross liver images, as well as H&E (hematoxylin-eosin) and Ki67 IHC staining images. Images were representative shown out of 6 independent mice per group. Scale bar, 200 μm. Source data are provided as a Source Data file.