Fig. 2: β-Catenin regulates the expression of SLC13A3 and intracellular SLC13A3 substrates.

a, b mRNA expression of TBX3, GLUL, and SLC13A3, as well as protein levels of β-catenin and SLC13A3 in CTNNB1-overexpressing or CTNNB1-knockdown HCC cells. Huh7 and HLF cells were transfected with pT3-EF1αH plasmid (empty vector, EV, gray) or pT3-EF1αH plasmid containing ΔN90-β-catenin mutant fragment (CTNNB1, red). HepG2 and SNU398 cells were infected with shRNA lentivirus using pLKO.1 plasmid containing either scramble shRNA (negative control shRNA, shNC, gray) or shCTNNB1 (blue) sequences. Cells were collected at 24 h for qRT-PCR and 48 h for western blot. The experiments were performed three times on different days. Each western blot represented one biological replicate (two technical repeats per group). Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. c Luciferase reporter assay for the identification of β-catenin binding sites in the SLC13A3 gene promoter region (~1.0 kb from transcription start site, TSS). A series of fragments in the SLC13A3 promoter region were schematized. HEK293T cells were transfected with the respective promoter plasmid, pCMV-renilla, and EV- or CTNNB1-overexpressing plasmid for 24 h. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. d Chromatin immunoprecipitation (ChIP)-PCR detection of the SLC13A3 promoter. DNA was isolated by anti-β-catenin antibody (orange), anti-TCF4 antibody (blue), or negative control IgG (gray). Input DNA which equaled 10% total DNA samples prior to immunoprecipitation was used as positive control. DNAs were respectively amplified using SLC13A3 promoter primers#1 (for binding site 1), #2 (for binding site 2), and #3 (spans −2100 to −2081 bp upstream of the TSS), as well as negative GAPDH primers and positive MYC primers. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. e In vitro EMSA analysis of TCF4 protein binding with two putative β-catenin binding sites in HepG2 nuclear extracts. The protein and DNA interactions were abolished by adding unlabeled wild-type probes, but could not be abrogated by mutant probes. Left panel: Adding anti-TCF4 antibody resulted in a supershift band to TCF4 protein. Right panel: No supershift band after adding anti-TCF4 antibody. SLC13A3 probe #1 was for the bind site 1, and SLC13A3 probe #2 was for the bind site 2. Each experiment was independently repeated three times. f Relative intracellular levels of malate, succinate, and fumarate. The data were obtained from the untargeted metabolomics in CTNNB1-overexpressing Huh7 cells (red) and CTNNB1-knockdown HepG2 cells (blue). Data are presented as the mean ± SEM (n = 6 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. Source data are provided as a Source Data file.