mouse hcc cell line hepa1 6  (ATCC)

Journal: Science Advances

Article Title: SLC13A2-transported citrate remodels transcriptional regulation through protein acetylation to suppress tumor growth

doi: 10.1126/sciadv.aec4368

Figure Lengend Snippet: ( A ) Liver tumor tissues were collected from the HTVi model and clinical patients and subjected to untargeted metabolomic profiling for pathway enrichment. ( B ) Summarized SLC transporters for TCA cycle intermediates. ( C ) Forest plot showing the hazard ratios (HR) for multiple genes encoding SLC transporters of TCA cycle intermediates in HCC. The horizontal line represents the 95% confidence interval. ( D ) Heatmap of detectable SLC transporters for TCA cycle intermediates in heterogeneous primary liver cancer models generated by genome editing of cancer driver genes selected by mutational frequency from human HCC cohorts (PRJNA674008). ( E ) qPCR analysis to screen candidate SLC transporters for HCC progression based on the relative expression of SLC transporters in the HTVi model ( n = 4 for the control group and n = 5 for the model group), mouse HCC Hepa1-6 cells, AML12 cells, and MPHs ( n = 3 independent experiments). ( F ) Venn diagram showing the overlap of significantly differentially expressed SLC transporters in the HTVi model, mouse HCC Hepa1-6 cells, and normal hepatocytes (AML12 and MPHs). ( G and H ) SLC13A2 expression in liver tissues from HTVi, AAV-cMYC/nRAS, and STZ-HFD HCC model. The data are presented as means ± SEM. * P < 0.05; ** P < 0.01, two-tailed unpaired Student’s t test. Ctrl, control; n.d., not determined; NS, not significant.

Article Snippet: The mouse HCC cell line Hepa1-6 and mouse normal hepatocyte cell line AML12 were obtained from the American Type Culture Collection as described previously ( ).

Techniques: Metabolomic, Generated, Expressing, Control, Two Tailed Test

Journal: Science Advances

Article Title: SLC13A2-transported citrate remodels transcriptional regulation through protein acetylation to suppress tumor growth

doi: 10.1126/sciadv.aec4368

Figure Lengend Snippet: ( A ) Relative abundance of TCA cycle and glycolysis metabolites in Hepa1-6 cells overexpressing SLC13A2 for 60 hours ( n = 3). ( B and C ) Mitochondrial respiration (OCR) and glycolysis (ECAR) measured by Seahorse XF analysis. ( D ) Untargeted metabolomics of vector- or SLC13A2-transfected Hepa1-6 cells cultured with [U- 13 C 6 ] glucose for 1 or 2 hours ( n = 3). ( E ) Relative abundance of pyruvate ( n = 3). ( F ) Cell viability/colony formation with the treatment of 500 μM pyruvate. * P < 0.05 versus vector without pyruvate; # P < 0.05 versus SLC13A2 without pyruvate. ( G ) Intracellular fractional labeling of citrate (m + 6) and acetyl-CoA (m + 2) in vector- or SLC13A2-transfected Hepa1-6 after 1 hour [U- 13 C 6 ] citrate tracing ( n = 3). ( H and I ) Relative abundance of oxaloacetate and extracellular/mitochondrial citrate levels ( n = 3). ( J ) Cell viability after 72 hours ACLY inhibitor (ACLYi) treatment (50 μM). * P < 0.05 versus vector without ACLY inhibitor; # P < 0.05 versus SLC13A2 without ACLY inhibitor. ( n = 4). ( K ) PKM2 subcellular protein levels after 60 hours transfection. ( L ) Co-IP assay showing the acetylation of PKM2. ( M ) PKM2 protein levels in SLC13A2-KD Hepa1-6 cells expressing WT/mutant PKM2, cultured for 60 hours. ( N ) Pyruvate kinase activity after 60 hours transfection ( n = 3). ( O ) SLC13A2 and/or PKM2 overexpression efficiency (left) and cell growth curves measured at 0, 24, 72, and 120 hours (right). * P < 0.05 SLC13A2 versus vector; # P < 0.05 SLC13A2 + PKM2 versus SLC13A2. ( P and Q ) Pyruvate kinase activity ( n = 10), pyruvate content ( n = 10), and PKM2 protein levels ( n = 6) in HTVi liver tissues with SLC13A2 LKO or OE. ( R ) NADH/NAD + ratio in HTVi liver tissues with SLC13A2 overexpression ( n = 6). Data are presented as means ± SEM. * P < 0.05, two-tailed unpaired Student’s t test [(A), (D), (E), (G) to (I), (N), and (P) to (R)]; two-way analysis of variance (ANOVA), followed by Bonferroni multiple comparisons [(F), (J), and (O)]. h, hour; IB, immunoblot; NC, Negative Control.

Article Snippet: The mouse HCC cell line Hepa1-6 and mouse normal hepatocyte cell line AML12 were obtained from the American Type Culture Collection as described previously ( ).

Techniques: Plasmid Preparation, Transfection, Cell Culture, Labeling, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Activity Assay, Over Expression, Two Tailed Test, Western Blot, Negative Control

Journal: Science Advances

Article Title: SLC13A2-transported citrate remodels transcriptional regulation through protein acetylation to suppress tumor growth

doi: 10.1126/sciadv.aec4368

Figure Lengend Snippet: ( A ) Total protein acetylation in liver tissues from HTVi mice with liver-specific overexpression or KO of SLC13A2 ( n = 3). ( B ) Immunoblot of subcellular fractions from Hepa1-6 cells overexpressing SLC13A2, probed with pan-acetyl-lysine antibody. ( C ) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of proteins lacking detectable acetylation sites identified by acetyl-proteomics in SLC13A2-OE Hepa1-6 cells. ( D ) GO molecular function enrichment analysis of proteins with detectable acetylation sites identified by acetyl-proteomics in SLC13A2-OE Hepa1-6 cells. ( E ) Histone modification levels in HTVi liver tumor tissues of control and SLC13A2 LKO mice detected by immunoblotting. Data are presented as means ± SEM ( n = 3). * P < 0.05; ** P < 0.01, versus controls, two-tailed unpaired Student’s t test. ( F ) Immunoblot analysis of histone acetylation marks in Hepa1-6 cells overexpressing SLC13A2. The data are representative from three independent experiments. ( G ) Immunoblot analysis of histone acetylation marks in Hepa1-6 cells transfected and treated with increasing concentrations of citrate (0, 250, and 500 μM) for 48 hours. ( H ) Volcano plot of hepatic genes in SLC13A2-OE mice. ( I ) Heatmap of representative genes involved in cell proliferation and metabolism. ( J ) GSEA of RNA-seq showing gene enrichment in SLC13A2-OE mice. ( K ) Heatmap of key TFs in SLC13A2-OE mice. NES, Normalized Enrichment Score.

Article Snippet: The mouse HCC cell line Hepa1-6 and mouse normal hepatocyte cell line AML12 were obtained from the American Type Culture Collection as described previously ( ).

Techniques: Over Expression, Western Blot, Modification, Control, Two Tailed Test, Transfection, RNA Sequencing

Journal: Science Advances

Article Title: SLC13A2-transported citrate remodels transcriptional regulation through protein acetylation to suppress tumor growth

doi: 10.1126/sciadv.aec4368

Figure Lengend Snippet: ( A ) Metagene and heatmap analyses showing the distribution of acetylated lysine (Acetyl-Lys) peaks around TSS (±2 kb) from ChIP-seq of Acetyl-Lys in SLC13A2-OE Hepa1-6 cells. ( B ) Circular chord diagram displaying GO enrichment of DEGs categorized by functional terms. ATPase, adenosine triphosphatase. ( C ) Bar plots of GO enrichment analysis for up-regulated (orange) and down-regulated (blue) genes from RNA-seq of SLC13A2-OE mice. ( D ) Representative IGV tracks showing ChIP-seq (Acetyl-Lys) signals in SLC13A2-OE Hepa1-6 cells and RNA-seq signals from liver tumor tissues of SLC13A2-OE mice at selected loci ( Igfbp6 and Apoa2 ). ( E ) IGV tracks of Acetyl-Lys ChIP-seq signals at Cebpa , Tcf21 , and Bhlhe41 loci, showing increased acetylation. ( F ) Public ChIP-seq tracks (H3K9ac, H3K27ac, and H4K12ac) from H1 human embryonic stem cells and HepG2 cells (ENCODE database) showing promoter acetylation at Cebpa , Tcf21 , and Bhlhe41 . ( G ) GO enrichment analysis of down-regulated DEGs from RNA-seq that are potential downstream targets of TFs Cebpa , Tcf21 , and Bhlhe41 . ( H ) Heatmap of representative downstream genes of Cebpa , Tcf21 , and Bhlhe41 in SLC13A2-OE tumor tissues.

Article Snippet: The mouse HCC cell line Hepa1-6 and mouse normal hepatocyte cell line AML12 were obtained from the American Type Culture Collection as described previously ( ).

Techniques: ChIP-sequencing, Functional Assay, RNA Sequencing