hepa1 6  (ATCC)

Journal: bioRxiv

Article Title: Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

doi: 10.64898/2026.05.08.723843

Figure Lengend Snippet: (A) Schematic of MG3-6_3-8 ABE architectures and comparison of terminal versus inlaid MG68-4 deaminase fusion strategies. Two copies of the MG68-4 adenosine deaminase were fused to the MG3-6_3-8 nickase either at the N- or C-terminus or inlaid at multiple internal positions. The residue numbering is relative to the MG3-6_3-8 nuclease. Bar plots show A→G editing efficiencies at individual adenines (A5–A10) within a representative target sequence. Boxplots depicting (B) Mean observed editing and (C) Max observed editing by individual ABE variants to oligomeric variants of 15 distinct gRNA targeting 2 different genes in Hepa1-6 cells. Editing data corresponding to n= 2 biologically independent replicates for each target guide. Individual data points represent each unique target’s locus.

Article Snippet: Hepa1-6 (ATCC #CRL-1830) and K562 (ATCC #CCL-243) cells were cultured in IMDM + GlutaMAX (Corning) supplemented with 10% FBS (Corning) for 1–2 passages prior to nucleofection.

Techniques: Comparison, Residue, Sequencing

Journal: bioRxiv

Article Title: Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

doi: 10.64898/2026.05.08.723843

Figure Lengend Snippet: (A) Schematic of the mammalian fluorescence-based screening assay. HEK293T cells were co-transfected with an ABE expression plasmid (pEditor, ABE-T2A-GFP), a reporter plasmid containing a stop codon–interrupted mCherry cassette (pReporter, BFP-target-stop-mCherry), and a targeting sgRNA. Cells were analyzed by flow cytometry three days post-transfection. Dual GFP/BFP gating was used to identify successfully transfected cells, and ABE activity was quantified by the fraction of mCherry-positive cells. ABE converts the target adenine in an in-frame stop codon of the target (B) mApoA1 in Hepa1-6 cells or (D, E) AAVS1 in HEK293T cells to a TGG sense codon, which enables expression of the mCherry protein. (C) Frequency of recovered MG3-6_3-8 ABE variants from the selection scheme with and without guide RNA, demonstrating guide-dependent enrichment of high-activity deaminase variants.

Article Snippet: Hepa1-6 (ATCC #CRL-1830) and K562 (ATCC #CCL-243) cells were cultured in IMDM + GlutaMAX (Corning) supplemented with 10% FBS (Corning) for 1–2 passages prior to nucleofection.

Techniques: Fluorescence, Screening Assay, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Activity Assay, Selection

Journal: The Journal of Clinical Investigation

Article Title: THEMIS attenuates MASH by suppressing disease-associated hepatocyte induction and hepatocyte senescence in mice

doi: 10.1172/JCI199303

Figure Lengend Snippet: ( A ) Immunoblotting of total lysates from WT and Themis -KO livers. ( B ) Immunoblotting of total lysates from Themis Flox and HKO livers. ( C ) Immunoblotting of total lysates from mouse and human primary hepatocytes transduced with Ad-GFP or Ad-Themis. ( D ) Immunoblotting of Themis Flox and HKO mice primary hepatocytes stimulated with 100 ng/mL EGF at indicated time points. ( E ) RAS activity assessment of EGF-treated primary hepatocytes isolated from Flox and HKO mice fed MASH diet for 5 months. ( F and G ) Hepa1 cells overexpressing either GFP or Themis were treated with 50 nM doxorubicin (Dox) for 3 days. ( F ) SA-β-GAL staining of Hepa1 cells. ( G ) Immunoblotting of total lysates from Hepa1 cells. ( H ) Immunoblotting of total lysates. Hepa1 cells overexpressing either GFP or Themis were treated with 1 μM Dox for 2 hours, followed by 10 nM MEK inhibitor trametinib treatment for 3 days. Scale bars: 50 μm ( F ).

Article Snippet: Hepa1 cells (ATCC, CRL-1830) were transduced by adenovirus and then treated with Dox (Cayman, 15007), MEK inhibitor trametinib (MedChemExpress, HY-10999)], or the combination.

Techniques: Western Blot, Transduction, Activity Assay, Isolation, Staining

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative Western blot analysis of GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells ( n = 3 independent experiments). ( B ) Quantitation of relative GPC3 protein level from (A). Data are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test. ( C ) Flow cytometry analysis of surface GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells. ( D ) Representative IHC images of GPC3 expression (brown) in tumor tissues from orthotopic Hepa1-6 tumor-bearing mice. Scale bar, 50 μm. ( E ) CLSM images of HepG2 and Hepa1-6 cells treated with SPD1 or SPD2 nanoparticles (50 μM; red fluorescence) for 6 hours. Scale bars, 20 μm. ( F ) Time-dependent CLSM imaging of HepG2 cells treated with SPD1 nanoparticles (50 μM) showing membrane-localized fibrillar transformation. Scale bars, 20 μm. ( G ) CLSM analysis of HepG2 cells sequentially incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours; red) and FITC-labeled anti-GPC3 antibody (green; 1:200; Abcam, #ab207080). Colocalization (yellow) indicates specific binding of SPD1 to membrane-bound GPC3. Fluorescence intensity and colocalization were quantified using MATLAB. Data are presented as means ± SD ( n = 3); n.s., not significant (one-way ANOVA with Tukey’s post hoc test). Scale bars, 20 μm. ( H ) SEM images of untreated HepG2 and WRL-68 cells or incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours). Magnified insets highlight membrane-associated fibrillar structures. ( I ) TEM images of untreated HepG2 cells (top) and those treated with SPD1 nanoparticles (50 μM, 24 hours; bottom). Red arrows indicate membrane-associated nanofibers. Scale bars, 500 nm. ( J ) SEM images showing the persistence of SPD1-derived fibrillar networks on HepG2 cells at 6, 24, and 72 hours posttreatment (50 μM). Scale bars, 2 μm. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: Human HepG2 (ATCC HB-8065), human WRL-68 (ATCC CL-48), and mouse Hepa1-6 (ATCC CRL-1830) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, A3161001C) and 1% penicillin/streptomycin (Beyotime, C0222) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Western Blot, Expressing, Quantitation Assay, Flow Cytometry, Fluorescence, Imaging, Membrane, Transformation Assay, Incubation, Labeling, Binding Assay, Derivative Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative CLSM images of HepG2 cells incubated with SPD1 nanoparticles (red, 50 μM) for 6 hours, followed by treatment with FITC-N 3 (10 to 50 μM, green) for an additional 6 hours. Merged yellow fluorescence indicates successful copper-free click conjugation between DBCO and N 3 on the cell membrane. Cells treated with SPD2+FITC-N 3 (50 μM) served as the nontargeted controls, showing minimal colocalization. Scale bars, 20 μm. ( B ) Time-dependent kinetics of bioorthogonal conjugation quantified by BCA protein assay and ICP-MS. HepG2 cells were pretreated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours), followed by incubation with Gd-DOTA-N 3 (50 μM) for 0.5, 1, 6, or 12 hours. Cells treated with Gd-DOTA-N 3 alone served as baseline controls. ( C ) T 1 -weighted MR images of HepG2 cells treated with Gd-DOTA (50 μM), Gd-DOTA-N 3 (50 μM), or sequentially with SPD1 or SPD2 (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( D ) Quantitative r 1 relaxivity under the corresponding treatment conditions in (C). ( E ) r 1 relaxivity of HepG2 cells preblocked with anti-GPC3 antibody (5 μg/ml, 12 hours; Abcam, #ab207080) before SPD1 treatment (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( F to H ) Cell viability of WRL-68 (F), HepG2 (G), and Hepa1-6 (H) cells after sequential treatment with SPD1 or SPD2 for 6 hours followed by Gd-DOTA-N 3 (50 μM, 6 hours). Cell viability was quantified using the CCK-8 assay. Data are presented as means ± SD { n = 3 for [(A) to (E)]; n = 6 for [(F) to (H)]}. Statistical significance was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant; n.s., not significant. All experiments were independently repeated three times with consistent results.

Article Snippet: Human HepG2 (ATCC HB-8065), human WRL-68 (ATCC CL-48), and mouse Hepa1-6 (ATCC CRL-1830) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, A3161001C) and 1% penicillin/streptomycin (Beyotime, C0222) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Incubation, Fluorescence, Conjugation Assay, Membrane, Bicinchoninic Acid Protein Assay, CCK-8 Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Pharmacokinetic profiles of SPD1 [10 mg/kg, intravenously (iv)] and SPD2 (10 mg/kg, iv) in healthy mice. Plasma concentrations were determined via PpIX fluorescence, and pharmacokinetic parameters were calculated using PKSolver 2.0 software. Data are expressed as means ± SD ( n = 3). ( B ) Blood Gd 3+ levels measured by ICP-MS in orthotopic Hepa1-6 tumor-bearing mice after Gd-DOTA-N 3 administration (0.1 mmol/kg, iv), with or without 6 hours pretreatment with SPD1 or SPD2 (10 mg/kg, iv). ( C ) Ex vivo fluorescence (FL) images of the tumors (T), heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (K), and intestine (I), at indicated time points following SPD1 injection (10 mg/kg, iv) in orthotopic Hepa1-6 tumor-bearing mice. Red dashed circles indicate tumors. Representative images from three mice per time point are shown. ( D ) Quantification of the fluorescence intensity in tumor and major organs of (C). ( E ) Representative micrographs demonstrate H&E staining, GPC3 IHC, and TUNEL staining in orthotopic Hepa1-6 tumor specimens from C57BL/6 mice treated with the following regimens: SPD1 pretreatment followed by Gd-DOTA-N 3 6 hours later, SPD2 pretreatment followed by Gd-DOTA-N 3 6 hours later, Gd-DOTA-N 3 alone, and Gd-DOTA alone. Representative images are shown from three biologically independent experiments. Scale bars, 100 μm. ( F ) Schematic illustration of the molecular imaging mechanism of the SPD1+Gd-DOTA-N 3 probe, highlighting self-assembly into nanofibers and signal amplification via r 1 enhancement. d, days. ( G ) In vivo T 1 -weighted images of subcutaneous Hepa1-6 tumor-bearing mice at multiple time points under four different experimental protocols. Red circles mark tumor regions. Representative images from three mice per group are shown. ( H ) In vivo T 1 -weighted images of orthotopic Hepa1-6 tumor-bearing mice under four different treatment protocols. Red circles mark tumor regions. Representative images from three mice per group are shown.

Article Snippet: Human HepG2 (ATCC HB-8065), human WRL-68 (ATCC CL-48), and mouse Hepa1-6 (ATCC CRL-1830) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, A3161001C) and 1% penicillin/streptomycin (Beyotime, C0222) at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Clinical Proteomics, Fluorescence, Software, Ex Vivo, Injection, Staining, TUNEL Assay, Imaging, Amplification, In Vivo

Journal: Frontiers in Immunology

Article Title: Targeting the TIGIT/CD155-induced metabolic checkpoint in NK cells restores anti-tumor immunity and suppresses hepatocellular carcinoma growth

doi: 10.3389/fimmu.2026.1790174

Figure Lengend Snippet: Validation of CD155-TIGIT signaling in SHP-2 recruitment and STAT3 pathway inhibition. (A) Schematic representation of the regulatory mechanism by which tumor cell CD155 modulates the SHP-2/STAT3 axis via TIGIT; (B) WB analysis of CD155 protein expression levels in Hepa1–6 cells; (C) Immunofluorescence staining showing SHP-2 expression and localization in co-cultured NK cells, scale bar: 25 µm; (D) Quantitative analysis of immunofluorescence signal intensity from panel C; (E) Co-IP assay demonstrating the interaction between SHP-2 and STAT3 in TIGIT-overexpressing NK cells; (F) Immunofluorescence colocalization analysis illustrating the distribution of SHP-2 and STAT3 in TIGIT-overexpressing NK cells, scale bar: 25 µm; (G) WB analysis of SHP-2, total STAT3, and its phosphorylated form p-STAT3 (Tyr705) protein levels in TIGIT or SHP-2-overexpressing NK cells. Experiments were conducted in triplicate. * indicates a statistically significant difference between groups, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The murine HCC cell line Hepa1-6 (ATCC ® CRL-1830TM) was maintained in high-glucose DMEM supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Biomarker Discovery, Inhibition, Expressing, Immunofluorescence, Staining, Cell Culture, Co-Immunoprecipitation Assay

Journal: Frontiers in Immunology

Article Title: Targeting the TIGIT/CD155-induced metabolic checkpoint in NK cells restores anti-tumor immunity and suppresses hepatocellular carcinoma growth

doi: 10.3389/fimmu.2026.1790174

Figure Lengend Snippet: Regulation of NK cell cytotoxicity against tumor cells via the TIGIT/STAT3/GLUT1 pathway. (A) Schematic diagram illustrating the modulation of NK cell anti-tumor activity through interventions on the TIGIT-STAT3-GLUT1 axis; (B, D) flow cytometry combined with CFSE/PI staining to assess the death rate of Hepa1–6 hepatocarcinoma cells, evaluating NK cell cytotoxicity; (C, E) LDH release assay measuring cell lysis levels in co-culture systems. Experiments were repeated three times. ** indicates p < 0.01 compared to the Control group, *** p < 0.001; # indicates p < 0.05 compared to the anti-TIGIT or IL-6+sh-NC groups.

Article Snippet: The murine HCC cell line Hepa1-6 (ATCC ® CRL-1830TM) was maintained in high-glucose DMEM supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Staining, Lactate Dehydrogenase Assay, Lysis, Co-Culture Assay, Control