human hepg2 cells  (ATCC)

Journal: Oncology Reports

Article Title: Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling

doi: 10.3892/or.2026.9074

Figure Lengend Snippet: Transcriptome analysis reveals the mechanisms underlying the enhanced efficacy of Regorafenib and Nifuroxazide in HCC. (A) Gene expression heatmap of key genes (including Vegfa, Bax and STAT3) in Regorafenib-treated compared with control HCC cells. (B) GSEA-based enrichment of Hallmark pathways in Regorafenib-treated HCC cells. (C) GSEA enrichment plot for the Hallmark_IL6_Jak_STAT3_Signaling pathway. (D) Representative results of the CCK-8 assay (for cell viability) of HepG2 cells under different treatments. (E) Statistical analysis of cell viability in . (F) Representative colony formation assay images of HepG2 cells under different treatments. (G) Statistical analysis of the number of colonies in . (H) Representative wound-healing assay images (0, 24 and 48 h) of HepG2 cells under different treatments. (I) Statistical analysis of wound closure rate in . Data are presented as mean ± standard deviation (n=3). *P<0.05 vs. the Control group; # P<0.05 vs. the Regorafenib group; $ P<0.05 vs. the Nifuroxazide group. HCC, hepatocellular carcinoma; GSEA, Gene Set Enrichment Analysis.

Article Snippet: Human HepG2 cells were authenticated by short tandem repeat (STR) profiling, which matched the reference STR data of ATCC HB-8065.

Techniques: Gene Expression, Control, CCK-8 Assay, Colony Assay, Wound Healing Assay, Standard Deviation

Journal: Oncology Reports

Article Title: Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling

doi: 10.3892/or.2026.9074

Figure Lengend Snippet: Effects of different treatments on apoptosis and the expression of related proteins. (A) Effects of Nifuroxazide combined with Regorafenib on HepG2 cell apoptosis detected by flow cytometry. (B) Statistical analysis of . (C) Expression of related proteins in cells detected by western blotting. (D) Statistical analysis of . Data are presented as mean ± standard deviation (n=3). *P<0.05 vs. the Control group; # P<0.05 vs. the Regorafenib group; $ P<0.05 vs. the Nifuroxazide group. STAT3, signal transducer and activator of transcription 3; PD-L1, programmed death ligand 1; VEGF, vascular endothelial growth factor; p-, phosphorylated.

Article Snippet: Human HepG2 cells were authenticated by short tandem repeat (STR) profiling, which matched the reference STR data of ATCC HB-8065.

Techniques: Expressing, Flow Cytometry, Western Blot, Standard Deviation, Control

Journal: Nucleic Acids Research

Article Title: RAPseq enables large-scale identification of RBP–RNA interactions and reveals essentials of post-transcriptional gene regulation

doi: 10.1093/nar/gkag090

Figure Lengend Snippet: RAPseq captures RBP-binding facilitated by RNA structure, sequence, and modification. ( A ) Schematic presentation of the experimental setup of RAPseq. RAPseq substrates are generated from native total RNA (left). Production of an RBP-Halo fusion allows protein purification and RNA-binding assay. Next-generation sequencing serves as readout using UMIs (right). ( B ) The dot plot displays binding of HUR to non-targeting (NT) and targeting (T1 and T2) RNA regions ( x -axis) of fragmented (left) and full-length (right) RNA from HepG2 cells. RAP-qPCR (left) was performed in two replicates (colored dots), and means (black horizontal lines) are shown. RNA-binding is displayed as log 2 -scaled fold change (∆∆Ct) of HUR over the -HaloTag control ( y -axis). RAPseq coverage tracks (right) represent HUR-binding to fragmented RNA [in reads per million (RPM)]. Genomic locations of the corresponding target regions (T1 and T2) are specified. Tracks for two HUR replicates and two RAPseq controls (HaloTag and RNA input) are shown. ( C–E ) Genome tracks demonstrate binding of (C) HUR to ARE and GRE motifs, (D) IRP1 to the iron-responsive element, and (E) YTHDF1 to a modified nucleotide (m 6 A) within KDELR2, FTL , and HNRNPA0 mRNAs, respectively, in HepG2 cells. Genomic locations with a scale bar indicate the length of the genomic region in bases, and gene features (black rectangle, exon and UTR; gray line, intron; arrow, direction of transcription) ( x -axis) and normalized read density (RPM, y -axis) are shown. Vertical lines highlight bound RNA elements. Tracks for two RBP replicates and m 6 A-specific RNA immunoprecipitation (IP, purple) over input control (green) are presented.

Article Snippet: HepG2 cells were obtained from the American Type Culture Collection (ATCC) with a certified genotype.

Techniques: Binding Assay, Sequencing, Modification, Generated, Protein Purification, RNA Binding Assay, Next-Generation Sequencing, Control, RNA Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: RNF90 promotes hepatic steatosis by degrading CPT1α to suppress fatty acid oxidation

doi: 10.1016/j.jbc.2026.111231

Figure Lengend Snippet: KLF5 mediates RNF90 expression upregulation in the liver of hepatic steatosis . A , representative H&E and Oil red O staining images of liver tissues from mice fed the indicated diets (n = 5). B – C , western blot ( B ) and quantitative analysis ( C ) of RNF90 and KLF5 protein expression in liver tissues from the mice described in ( A ) (n = 4). D , RT-qPCR analysis of RNF90 mRNA expression in mouse liver tissues (12 W NCD, HFD: n = 8; ob/ob: n = 6; 19 W NCD, HFHC: n = 10). E – H , RT-qPCR ( E , G ; n = 4) and western blot ( F , H ; n = 3) analyses of the indicated genes’ expressions in POA-treated HepG2 and AML12 cells at different time points. I , schematic of RNF90 gene promoter regions analysis via NCBI’s in silico tools. J , predicted conserved KLF5 binding sites via the JASPAR database. K – L , analysis of RNF90 mRNA ( K ; n = 4) and protein ( L ; n = 3) expression in HEK293T cells with KLF5 overexpression ( left panel ) and KLF5 knockdown ( right panel ). M , dual-luciferase reporter assay analysis of RNF90 promoter activity in KLF5-overexpressed ( left panel ) or KLF5-knockdown ( right panel ) HEK293T cells (n = 4). N , dual luciferase reporter assay for analyzing the effect of KLF5 on truncated RNF90 promoter mutants in HEK293T cells (n = 4). O , ChIP-PCR analysis of KLF5 enrichment at its potential binding sites in RNF90 promoter in POA-treated HepG2 cells (n = 3); schematic shows ChIP-PCR primer positions ( black arrows ). Data are presented as the mean ± SD, and statistical significance was analyzed by two-tailed t test ( C , N , and O ) and one-way ANOVA followed by Tukey’s post hoc test ( D , E , G , K , and M ). The exact p values are shown in each graph. ChIP-PCR, chromatin immunoprecipitation PCR; HFD, high-fat diet; HFHC, high-fat, high fructose and high-cholesterol; KLF5, Kruppel-like factor 5; NCBI, National Center for Biotechnology Information; NCD, normal chow diet; POA, a mixture of palmitic acid and oleic acid; RT-qPCR, quantitative real-time PCR.

Article Snippet: HEK293T and HepG2 cell lines were obtained from ATCC, and AML12 were kindly provided by Professor Han Yan (Zhejiang University).

Techniques: Expressing, Staining, Western Blot, Quantitative RT-PCR, In Silico, Binding Assay, Over Expression, Knockdown, Luciferase, Reporter Assay, Activity Assay, Two Tailed Test, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: RNF90 promotes hepatic steatosis by degrading CPT1α to suppress fatty acid oxidation

doi: 10.1016/j.jbc.2026.111231

Figure Lengend Snippet: RNF90 aggravates hepatic steatosis in vitro . A , RT-qPCR (n = 4) and western blot (n = 3) analyses of RNF90 mRNA and protein expression in indicated HepG2 stable cell lines. B – C , Oil red O staining ( B ) and TG content measurement ( C ) in BSA- or POA-treated RNF90-modified HepG2 stable cell lines (n = 4). D – E , validation of RNF90 knockdown efficiency at the mRNA ( D , n = 4) and protein levels ( E , n = 3) in AML12 cells stably expressing shRNF90 or control shRNA. F – G , Oil red O staining ( F ) and TG content measurement ( G ) in BSA- or POA-treated AML12 stable cell lines with stable RNF90 knockdown (n = 4). H – I , RT-qPCR ( H , n = 4) and western blot ( I , n = 3) analyses of RNF90 mRNA and protein expression in HepG2 and AML12 cells stably overexpressing RNF90 wildtype or its E3 ligase-deficient mutant (RNF90-DM). J – K , Oil red O staining ( J ) and TG content detection ( K ) in BSA- or POA-treated HepG2/AML12 cells stably overexpressing RNF90 wildtype or RNF90-DM cells (n = 4). Data are presented as the mean ± SD, and statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc test ( A , C , D , G , H , and K ). The exact p values are indicated in each graph. BSA, bovine serum albumin; POA, a mixture of palmitic acid and oleic acid; RT-qPCR, quantitative real-time PCR; shRNF90, shRNA targeting RNF90; TG, triglyceride.

Article Snippet: HEK293T and HepG2 cell lines were obtained from ATCC, and AML12 were kindly provided by Professor Han Yan (Zhejiang University).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Stable Transfection, Staining, Modification, Biomarker Discovery, Knockdown, Control, shRNA, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: RNF90 promotes hepatic steatosis by degrading CPT1α to suppress fatty acid oxidation

doi: 10.1016/j.jbc.2026.111231

Figure Lengend Snippet: RNF90 regulates CPT1α stability in an E3 ligase activity-dependent manner . A , immunoblotting analysis of CPT1α protein expression in HEK293T cells cotransfected with HA-CPT1α and increasing concentrations of Flag-tagged RNF90 wildtype (RNF90-WT) or its E3 ligase-deficient mutant (RNF90-DM) plasmids. B – D , immunoblotting ( B , C ) and densitometric quantification ( D ) analyses showing the effect of RNF90-WT or RNF90-DM on CPT1α protein half-life by CHX chase assay. E , immunoblotting analysis of CPT1α protein expression in HEK293T cells cotransfected with increasing amounts of RNF90 plasmid and either HA-tagged CPT1α-WT or HA-tagged ubiquitination-deficient CPT1α mutant (CPT1α-K195R) plasmids. F – G , immunoblotting ( F ) and quantification ( G ) analyses of CPT1α-WT or CPT1α-K195R protein half-life as regulated by RNF90 in HEK293T cells. H – I , immunoblotting ( H ) and quantification ( I ) analyses of RNF90 knockdown on CPT1α protein half-life in CHX-treated HEK293T cells. J , immunoblotting analysis to delineate the degradation pathway of CPT1α protein in HEK293T cells transfected with the indicated plasmids, and subsequently treated with CHX alone or in combination with MG132, CQ, or NH 4 Cl prior to cell harvest. K , western blot analysis of CPT1α protein expression in POA-treated HepG2 and AML12 cells, including RNF90-WT/DM-overexpressing stable lines, RNF90-knockdown stable lines, and their respective control cells. L , measurement of cellular FAO activity in BSA- or POA-treated HepG2 cells with stable RNF90 overexpression or knockdown. Data are presented as the mean ± SD, and statistical significance was analyzed by two-tailed t test ( D and G ) and one-way ANOVA followed by Tukey’s post hoc test ( I and L ). The exact p values are indicated in each graph. BSA, bovine serum albumin; CHX, cycloheximide; CPT1α, carnitine palmitoyltransferase 1α; CQ, chloroquine; FAO, fatty acid oxidation; POA, a mixture of palmitic acid and oleic acid.

Article Snippet: HEK293T and HepG2 cell lines were obtained from ATCC, and AML12 were kindly provided by Professor Han Yan (Zhejiang University).

Techniques: Activity Assay, Western Blot, Expressing, Mutagenesis, Plasmid Preparation, Ubiquitin Proteomics, Knockdown, Transfection, Control, Over Expression, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: RNF90 promotes hepatic steatosis by degrading CPT1α to suppress fatty acid oxidation

doi: 10.1016/j.jbc.2026.111231

Figure Lengend Snippet: CPT1α is essential for RNF90-mediated regulation of FAO and steatosis in hepatocytes . A , HepG2 cells stably overexpressing Flag-tagged RNF90 were transfected with either HA-tagged CPT1α plasmid or empty control plasmid. At 12 h post transfection, cells were treated with POA for 24 h, then harvested for immunoblotting analysis of Flag-tagged RNF90 and HA-tagged CPT1α protein expression, with β-actin as the loading control. B , measurement of FAO activity in HepG2 cells treated as described in ( A ) (n = 4). C – D , representative Oil red O staining images, quantitative analysis of Oil red O-positive area ( C ), and TG content quantification ( D ) in HepG2 cells treated as described in (A) (n = 4). E , HepG2-shRNF90-3# cells were transfected with shCPT1α plasmid or empty control plasmid. After 12 h of transfection, cells were treated with POA for 24 h, then harvested for immunoblotting analysis of RNF90 and CPT1α protein expression, with β-actin as the loading control. F , measurement of FAO activity in HepG2 cells treated as described in ( E ) (n = 4). G – H , representative Oil red O staining images, quantitative analysis of Oil red O-positive area ( G ) and TG content quantification ( H ) in HepG2 cells treated as described in ( E ) (n = 4). I , schematic diagram illustrating the underlying molecular mechanism by which the KLF5/RNF90/CPT1α axis promotes hepatic steatosis by suppressing fatty acid oxidation. Data are presented as the mean ± SD, and statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc test ( B – D , F – H ). The exact p values are indicated in each graph. CPT1α, carnitine palmitoyltransferase 1α; FAO, fatty acid oxidation; KLF5, Kruppel-like factor 5; POA, a mixture of palmitic acid and oleic acid; shRNF90, shRNA targeting RNF90; TG, triglyceride

Article Snippet: HEK293T and HepG2 cell lines were obtained from ATCC, and AML12 were kindly provided by Professor Han Yan (Zhejiang University).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Western Blot, Expressing, Activity Assay, Staining, shRNA

Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: Depletion of UFL1 causes aberrant oxidative stress and NRF2-ARE pathway activation. (A ) WikiPathways analysis of significantly differentially expressed genes in liver tissues from 3-month-old CKO vs. WT mice. This analysis revealed significant enrichment of several pathways associated with oxidative stress or metabolism. (B) Gene Set Enrichment Analysis (GSEA) of the Nrf2 pathway using RNA-seq data from liver tissues of 3-month-old CKO vs. WT mice. The normalized enrichment score (NES), nominal p-value, and false discovery rate (FDR) q-value were used to assess the significance of the association between gene sets and the signaling pathway. Criteria for statistical significance were set as |NES| > 1, FDR ≤0.25, and P < 0.05. (C) Expression levels of Nrf2 target genes in liver tissues were quantified by qPCR. Error bars represent the mean ± SD. ∗, P < 0.05; ∗∗, P < 0.01; ns, not significant. (D) Schematic of the NRF2-responsive reporter construct. Fluorescence intensity was visualized using fluorescence microscopy. Scale bars: 100 μm. (E) HepG2 cells transfected with the ARE-GFP reporter were verified by Western blot using the indicated antibodies. (F) Immunofluorescence staining for NRF2 (green) in cells following UFL1 knockdown or exogenous UFL1 rescue, respectively. Cell nuclei were counterstained with DAPI, and merged images show colocalization of NRF2 with DAPI. Scale bar: 50 μm. (G) Quantification of the NRF2 nuclear/cytoplasmic ratio. This ratio reflects the proportion of NRF2 protein localized in the nucleus relative to that in the cytoplasm, based on 30 randomly selected cells counted. Error bars represent the mean ± SD. ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, not significant.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Activation Assay, RNA Sequencing, Expressing, Construct, Fluorescence, Microscopy, Transfection, Western Blot, Immunofluorescence, Staining, Knockdown

Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: Loss of UFL1 leads to activation of the NRF2-ARE pathway in hepatic cells. (A) Western blot analysis of the expression of Keap1, Nrf2, p62, Nqo1, SLC7A11, HO-1, and Gpx4 proteins in the livers of 3-month-old WT and CKO mice. (B) Western blot analysis of the expression of KEAP1, NRF2, p62, UFL1, and DDRGK1 proteins in HepG2 cells with UFL1 knockdown or control cells. (C) Western blot analysis of the expression of KEAP1, NRF2, UFM1, and UFSP2 proteins in HepG2 cells with UFSP2 knockdown or control cells. (D) Western blot analysis of the expression and subcellular localization of NRF2 in HepG2 cells with UFL1 knockdown or control cells. Calnexin, Histone H3, and GAPDH were used as loading control, respectively. (E) Immunohistochemical staining of Nrf2 in liver sections from 3-month-old WT and CKO mice. The larger dashed-boxed images below their corresponding overview images in the Nrf2 staining panel show higher-magnification views of Nrf2 nuclear staining. Scale bars: 100 μm. (F) Quantification of the number of cells with Nrf2 nuclear localization from Nrf2 immunohistochemical staining in liver sections from 3-month-old WT and CKO mice. Quantification was carried out from three WT and three CKO mice, at least 400 cells counted per mouse. Error bars represent the mean ± SD. ∗∗, P < 0.01.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Control, Immunohistochemical staining, Staining

Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: UFMylation stabilizes KEAP1 by antagonizing its ubiquitination. (A) UFMylation assay of endogenous KEAP1 in HepG2 cells. HepG2 whole-cell lysates were subjected to co-immunoprecipitation (Co-IP) with KEAP1 (Ab) antibody or control IgG, followed by Western blot. Small black arrowheads indicate bands corresponding to covalently linked KEAP1-UFM1. (B, C) Analysis of the interaction between KEAP1 and UFL1 in HEK293T cells. HEK293T cell lysates were subjected to immunoprecipitation with anti-HA ( B ) or anti-FLAG ( C ) affinity gel, followed by Western blot to detect their mutual interaction. NRF2 and DDRGK1 served as positive controls for binding to KEAP1 and UFL1, respectively. (D) KEAP1 UFMylation was verified by Western blot following immunoprecipitation in UFSP1 KO /UFSP2 KO HEK293T cells expressing exogenous UFMylation system components and FLAG-tagged KEAP1. (E) KEAP1 ubiquitination was analyzed by Western blot in UFSP1 KO /UFSP2 KO HEK293T cells transfected with His-tagged ubiquitin (His-Ub) alone or with His-Ub plus UFMylation components. (F) KEAP1 protein stability was assessed by Western blot in UFL1-knockdown (sgUFL1) versus control HepG2 (sgCtrl) cells. Cells were treated with 100 μg/mL CHX for the indicated time. (G) Quantification of KEAP1 protein levels (corresponding to F ) is shown in the graph. Error bars represent the mean ± SD. ∗, P < 0.05.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Binding Assay, Expressing, Transfection, Knockdown

Journal: Biotechnology Reports

Article Title: Insulin Resistance, Anti-inflammatory, and Antioxidant In vitro Activity of Heterologously Expressed Arenin from Dryophytes arenicolor

doi: 10.1016/j.btre.2026.e00947

Figure Lengend Snippet: In vitro modulation of hepatic insulin resistance by arenin in hepatic insulin-resistant (HepG2) cells . Arenin concentrations (3.90–1000 µg/mL) were incubated for 5 h in hepatic insulin-resistant cells. Negative control (N) cells were cultured in the absence of insulin, whereas insulin-resistant (IR) control cells were cultured in the presence of insulin. Rosiglitazone (ROSI) at 10 μM (3.57 µg/mL) was used as the positive reference control. Different letters abc indicate statistical differences between concentrations analyzed using Tukey test ( p < 0.05).

Article Snippet: Human hepatocyte carcinoma (HepG2) cells, human primary dermal fibroblasts (HDFa) cells, and murine macrophage (RAW 264.7) cells were obtained from the American Type Culture Collection (ATCC, VA, USA).

Techniques: In Vitro, Incubation, Negative Control, Cell Culture, Control