Journal: Nature Communications
Article Title: SLC13A3 is a major effector downstream of activated β-catenin in liver cancer pathogenesis
doi: 10.1038/s41467-024-51860-2
Figure Lengend Snippet: 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, sh NC , gray) or sh CTNNB1 (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.
Article Snippet: The reaction mixtures (1 μg HepG2 nuclear protein, 1 pmol probes annealed on 95 °C for 5 min in a heat block, 1 μg poly d(I-C), 2 μL of 5X Binding Buffer, 1.0 µL of TF probe, 2.0 µL of cold TF probe for competitive group, 1.5 μL of anti-TCF4 antibody for supershift band) were incubated according to manufacturer’s protocol and then loaded on a 6% native polyacrylamide gel in 0.5% TBE buffer and blotted onto Biodyne™ B Nylon Membrane (0.45 μm, Thermo Fisher Scientific, Gaithersburg, MD, USA).
Techniques: Expressing, Knockdown, Transfection, Plasmid Preparation, Mutagenesis, Infection, shRNA, Negative Control, Quantitative RT-PCR, Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Binding Assay, Chromatin Immunoprecipitation, Isolation, Immunoprecipitation, Positive Control, Amplification, In Vitro, Protein Binding
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![The variant rs3826795 at 19q13.3 regulates transcriptional activity of HIF3A by modulating the binding affinity of <t>VEZF1</t> to the promoter Allele-specific constructs containing the putative promoter sequence flanking rs3826795 were cloned into the pGL3-basic luciferase reporter vector and transfected into the (A) HEK-293, (B) HEPM, and (C) HOK. The results of luciferase activity were normalized to those of pGL3 basic ( n = 6). (D) EMSA with biotin-labelled oligonucleotides containing the rs3826795[A] or rs3826795[G] allele and nuclear extracts from HEPM and HOK. Lanes 1 showed the mobility of the labeled oligonucleotides without nuclear extracts; lanes 2 and 4 showed the mobility of the labeled oligonucleotides with nuclear extracts in the absence of the competitor oligonucleotide and lanes 3 and 5 showed the mobility of the labeled oligonucleotides with nuclear extracts in the presence of unlabeled competitors. The arrow indicates a DNA–protein complex. (E) In silico predicted preferential binding of VEZF1 to the non-risk allele C of rs3826795. (F) The transcription factor VEZF1 was analyzed for correlation with Hif3a in mouse embryo craniofacial tissues, lip and palate tissues, palate shelves (from left to right). (G) The ChIP enrichment of VEZF1 as determined by ChIP-qPCR. IgG was used as a negative control. (H) <t>Supershift</t> assays using the VEZF1 antibody in HEPM and HOK. IgG was used as a negative control. The data are presented as the means ± SD, and the p values were calculated using Student’s t test. ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001, and ∗∗∗∗ indicates p < 0.0001. EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. SD, standard deviation.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4074/pmc11834074/pmc11834074__gr2.jpg)
