hcc 70 crl 2315 human breast cancer cell lines  (ATCC)

Journal: iScience

Article Title: Sunitinib induces immunogenic cell death through eIF2α phosphorylation to potentiate immunotherapy in HCC

doi: 10.1016/j.isci.2026.115378

Figure Lengend Snippet: Immune characteristics of the ICDRS model and repurposed drug discovery in HCC (A and B) Correlation of risk score in TCGA-LIHC with stromal, immune according to the ESTIMATE algorithm. (C) Correlation analysis between risk score and TMB. (D) Comparison of immune cell infiltration levels between ICDRS subtypes of TCGA-LIHC. ns , not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (E) The relative distribution of IPS identified by the status of CTLA-4 and PD-1 was compared between high-versus low-risk group in TCGA-LIHC cohort. (F) Correlation of TIDE status and ICD related risk signature in HCC patients. (G) Predictive results of TIDE algorithm on anti-PD-L1 response rates in HCC patients from TCGA-LIHC. (H) Drug candidates identified via cross-analysis of the CMap, L1000 FWD, and DGIdb databases were sunitinib, quercetin, and erythromycin. (I) Molecular docking of eIF2α (PDB ID: 8DYS ) with sunitinib showing the interaction site. (J) DARTs was performed in untreated Hepa1-6 cells lysates incubated with sunitinib at 50 μM.

Article Snippet: HCC cell lines (Hepa1-6, Hep3B, and MHCC-97H) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cat#11054001, Gibco) supplemented with 10% fetal bovine serum (FBS; Cat#FS201-02, TransGen) and 100 U/ml penicillin/streptomycin (Cat#15140122, Gibco) at 37°C under 5% CO 2 .

Techniques: Drug discovery, Comparison, Incubation

Journal: iScience

Article Title: Sunitinib induces immunogenic cell death through eIF2α phosphorylation to potentiate immunotherapy in HCC

doi: 10.1016/j.isci.2026.115378

Figure Lengend Snippet: Sunitinib suppresses HCC cell/organoid proliferation and induces ICD-associated DAMP release in vitro (A–C) A subsequent analysis of cell viability was conducted using CCK-8 following a time-dependent manner. Each panel displays the HCC cell lines (Hep3B, MHCC-97H, and Hepa 1–6) that were subjected to sunitinib treatment for 24 h, 48 h, and 72 h ( n = 3). (D) A clone formation assay was performed on HCC cell lines (MHCC-97H, Hep3B, and Hepa1-6) that were treated with different concentrations of sunitinib (0, 0.5, 1, 2.5, and 5 μM). ( n = 3). (E and F) The HCC organoid growth assay was treated with different concentrations of sunitinib (0, 0.5, 1, 5, and 10 μM). Scale bars: 1000 μm. The quantification of the results is shown in (F). ( n = 3). (G–I) ATP assay of HCC cell lines (MHCC-97H, Hep3B, and Hepa1-6) was treated with sunitinib at the concentrations and times shown in the figure. ( n = 3). (J) Signature proteins of ICD in two HCC cell lines were analyzed by Western blot assays. Cells were treated with sunitinib for 24 h for the analysis of membrane CRT and other proteins, while HMGB1 secretion was assessed in the supernatant collected after 48 h of treatment. The unlabeled CRT and HMGB1 signals originate from the cell membrane and the supernatant, respectively. ( n = 3). (K and L) Immunoblots and its quantitation of phosphorylated eIF2α ( p -eIF2α) and total eIF2α in Hepa1-6 cells treated by sunitinib for indicated time points. ( n = 3). (M) Detection of cell surface calreticulin (CRT) exposure by flow cytometry in HCC cells following 24-h sunitinib treatment. ( n = 3). Data are presented as means ± SD. Statistical analysis was performed using a Student’s t test (F–I and L). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: HCC cell lines (Hepa1-6, Hep3B, and MHCC-97H) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cat#11054001, Gibco) supplemented with 10% fetal bovine serum (FBS; Cat#FS201-02, TransGen) and 100 U/ml penicillin/streptomycin (Cat#15140122, Gibco) at 37°C under 5% CO 2 .

Techniques: In Vitro, CCK-8 Assay, Tube Formation Assay, Growth Assay, ATP Assay, Western Blot, Membrane, Quantitation Assay, Flow Cytometry

Journal: iScience

Article Title: Sunitinib induces immunogenic cell death through eIF2α phosphorylation to potentiate immunotherapy in HCC

doi: 10.1016/j.isci.2026.115378

Figure Lengend Snippet: Sunitinib induces ICD in HCC cell lines by facilitating eIF2α phosphorylation (A–D) Hepa 1–6 and Hep3B cells were treated with sunitinib (5 μM) for 6 h, with or without co-treatment with ISRIB (1 μM; added 2 h prior to sunitinib). Protein lysates were analyzed by immunoblot for the indicated proteins, with quantification shown in (B) and (D). ( n = 3). (E and F) Immunofluorescence cell-surface staining of CRT (green) in Hepa 1–6 and Hep3B treated for 24 h with sunitinib alone or in combination with 1 μM ISRIB. ( n = 3). Scale bars: 10 μm. (G–J) Flow cytometry was performed to analyze the surface expression of CRT in HCC cells treated with sunitinib for 24 h ( n = 3). Data are presented as means ± SD. Statistical analysis was performed using a Student’s t test (B, D, I, and J). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: HCC cell lines (Hepa1-6, Hep3B, and MHCC-97H) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cat#11054001, Gibco) supplemented with 10% fetal bovine serum (FBS; Cat#FS201-02, TransGen) and 100 U/ml penicillin/streptomycin (Cat#15140122, Gibco) at 37°C under 5% CO 2 .

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Staining, Flow Cytometry, Expressing

Journal: iScience

Article Title: Sunitinib induces immunogenic cell death through eIF2α phosphorylation to potentiate immunotherapy in HCC

doi: 10.1016/j.isci.2026.115378

Figure Lengend Snippet: Sunitinib in combination with anti-PD-L1 elicits synergistic anti-HCC efficacy (A) The scheme of tumor incubation and treatment approach. (B and C) Survival curves (B) and tumor growth (C) of Hepa1-6-induced subcutaneous HCC in C57BL/6 mice treated with vehicle or single agent or combination-treated Hepa1-6 cells. ( n = 8–10). (D) Mice were sacrificed at day 21 after treatment, tumor weight was measured. (E) Representative photographs of dissected tumors from each group. (F) Western blot analysis of p -eIF2α and eIF2α protein expression levels in tumors from C. ( n = 3). (G) Relative band density graphs for p -eIF2α normalized to eIF2α from F. (H–K) Flow cytometry plots of percentages of tumor-infiltrating CD45 + CD11c + DCs (H), CD8 + IFN-γ + T cells (I), CD11b + Gr-1 + MDSCs (J), and Foxp3 + CD4 + CD3 + T cells (K) within the tumors following the indicated treatment groups. ( n = 3). (L) Multiplex immunofluorescence staining of the markers shown in the diagram in Hepa1-6-induced subcutaneous HCC model. ( n = 3). Scale bars: 50 μm. Survival curves were generated using the Kaplan-Meier method, and statistical significance was assessed by the log rank test (B). Data in C was analyzed by two-way ANOVA. Comparisons between two groups (D and G–K) were performed using an unpaired two-tailed Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: HCC cell lines (Hepa1-6, Hep3B, and MHCC-97H) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cat#11054001, Gibco) supplemented with 10% fetal bovine serum (FBS; Cat#FS201-02, TransGen) and 100 U/ml penicillin/streptomycin (Cat#15140122, Gibco) at 37°C under 5% CO 2 .

Techniques: Incubation, Western Blot, Expressing, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, Generated, 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 ) 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