hepg2 cell extracts  (Aviva Systems)

Journal: Antioxidants

Article Title: Imeglimin Halts Liver Damage by Improving Mitochondrial Dysfunction in a Nondiabetic Male Mouse Model of Metabolic Dysfunction-Associated Steatohepatitis

doi: 10.3390/antiox13111415

Figure Lengend Snippet: Effect of imeglimin on palmitic acid-stimulated lipid accumulation and apoptosis in human hepatocyte. ( A ) Representative microphotographs of palmitic acid (PA)-stimulated HepG2 stained with Oil Red O. ( B , C ) Intracellular mRNA expression of ( B ) fatty acid (FA) oxidation-related genes and ( C ) lipogenesis-related genes in PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. ( D , E ) Intracellular level of ( D ) cleaved caspase-3 and ( E ) cleaved PARP in PA-stimulated HepG2. *: p < 0.05, **: p < 0.01, significant difference between groups by Student’s t -test. ( F ) Western blotting for phospho-JNK (p-JNK), total JNK (t-JNK), BAX and BCL-2 expression in PA-stimulated HepG2. ( G ) Intracellular level of TNF-α, IL-6 and IL-1β in PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. ( H ) Intracellular mRNA expression of ER stress-related genes in PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. ( I , J ) Intracellular ( I ) protein and ( J ) mRNA expression of profibrogenic markers in LX-2 co-cultured with PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. HepG2 cells were treated with 0.5 mM of PA and 0, 1, 3, 10 mM of imeglimin under both mono-culture and co-coculture. Gapdh and Actin were used as an internal control for qRT-PCR and western blotting, respectively. Quantitative values are indicated as fold changes to the values of PA (−)/Igm 0 mM-treated group ( B – E , H , J ). Data are the mean ± SD ( n = 8; ( B – E , G , H , J )). N.S, not significant.

Article Snippet: In vitro cell apoptosis in HepG2 cells was determined as cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) 1 concentration in cell extracts using ELISA and FastScanTM cleaved PARP (Asp214) ELISA kit (Cell Signaling Technology, Danvers, MA, USA), respectively, following the manufacturer’s instructions.

Techniques: Staining, Expressing, Western Blot, Cell Culture, Control, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Imeglimin Halts Liver Damage by Improving Mitochondrial Dysfunction in a Nondiabetic Male Mouse Model of Metabolic Dysfunction-Associated Steatohepatitis

doi: 10.3390/antiox13111415

Figure Lengend Snippet: Effect of imeglimin on mitochondrial dysfunction in palmitic acid-stimulated human hepatocyte. ( A ) Representative images of TMRM live stains corresponding to mitochondrial membrane potential in palmitic acid (PA)-stimulated HepG2. ( B ) Quantification of TMRM intensity per cell; data shown as the mean ± SD for 50 cells per condition in three representative experiments. Nuclei were stained with Hoechst 33342. **: p < 0.01, significant difference between groups by Student’s t -test. ( C ) Relative activity of mitochondrial respiratory chain complex I–V in PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. ( D ) Measurements of oxygen consumption rate (OCR) using a seahorse extracellular flux analyzer. ( E ) Calculations of the basal and maximal respiration rates. **: p < 0.01, significant difference between groups by Student’s t -test. ( F , G ) Intracellular ( F ) mRNA and ( G ) protein expression of mitochondrial biogenesis markers in PA-stimulated HepG2. **: p < 0.01, significant difference between groups by Student’s t -test. HepG2 cells were treated with 0.5 mM of PA and 0, 1, 3, 10 mM of imeglimin. Gapdh and Actin were used as an internal control for qRT-PCR and western blotting, respectively. Quantitative values are indicated as fold changes to the values of PA (−)/Igm 0 mM-treated group ( B , C , F ). Data are the mean ± SD ( n = 8; ( B – F )).

Article Snippet: In vitro cell apoptosis in HepG2 cells was determined as cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) 1 concentration in cell extracts using ELISA and FastScanTM cleaved PARP (Asp214) ELISA kit (Cell Signaling Technology, Danvers, MA, USA), respectively, following the manufacturer’s instructions.

Techniques: Membrane, Staining, Activity Assay, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of molecular endocrinology

Article Title: A 5′ distal palindrome within the mouse mammary tumor virus-long terminal repeat recruits a mammary gland-specific complex and is required for a synergistic response to progesterone plus prolactin

doi: 10.1677/JME-08-0027

Figure Lengend Snippet: Origin of cell lines used for screening of mammary gland-specific complex (MGSC) binding to the −941/−930 palindromic site

Article Snippet: The exceptions were nuclear extracts from HeLa, HepG2 and U-937 cells that were purchased from Active Motif (Carlsbad, CA, USA).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

doi: 10.1038/s41598-019-52837-8

Figure Lengend Snippet: Decreased GABPB1 expression levels during ferroptosis in HepG2 cells. ( A ) HepG2 cells were treated with erastin (10 μM) or ferrostatin-1 (1 μM) as indicated for 24 h, and cell viability was determined using CCK-8 assays. ( B , C ) HepG2 cells were treated with erastin (10 μM) for 24 h, and GABPB1 mRNA ( B ) and protein ( C ) levels were determined. ( D ) Schematic illustration of the genomic locus of GABPB1 potential binding sites in the PRDX5 promoter. ( E ) ChIP and RT-qPCR assays confirmed the binding loci of GABPB1 to the PRDX5 promoter. ( F ) HepG2 cells were treated with erastin (10 μM) for 24 h, and PRDX5 protein levels were determined. ( G , I ) Untransfected HepG2 cells or cells transfected as indicated were treated with 10 μM erastin for 24 h. ROS generation was determined by DCFH-DA staining, and cells were observed under a fluorescence microscope. GABPB1 ( H ) and PRDX5 ( J ) protein levels were determined. Bars: 50 μm. ( K , N ) ROS were measured in cells treated as indicated by flow cytometry using DCFH-DA staining. ( L , O ) MDA levels were measured. ( M , P ) Cell viability was assayed in cells treated as indicated. Values are expressed as the means ± SD (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: ChIP was performed on the chromatin extract from HepG2 cells according to the instructions provided by Merck-Millipore.

Techniques: Expressing, CCK-8 Assay, Binding Assay, Quantitative RT-PCR, Transfection, Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: Scientific Reports

Article Title: LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

doi: 10.1038/s41598-019-52837-8

Figure Lengend Snippet: Increased GABPB1-AS1 expression levels during ferroptosis in HepG2 cells. ( A ) Schematic illustration of the genomic locus of GABPB1, GABPB1-AS1 and two neighbouring genes on human chromosome 15q21.2. The GABPB1 and HDC genes are encoded by the (−) DNA strand, while the GABPB1-AS1 and USP8 genes are encoded by the (+) DNA strand. ( B , C ) GABPB1-AS1 was predicted to be a poor protein coding capacity. The RNA sequences of GABPB1, ACTB, GABPB1-AS1 and MALAT1 were evaluated by the CPC ( B ) and CPAT ( C ) programmes. MALAT1 served as a control noncoding transcript, while GABPB1 and ACTB served as control protein coding transcripts. ( D ) Subcellular localization of GABPB1-AS1, GAPDH and MALAT1. GAPDH mRNA was used as a control for the cytoplasmic fraction; MALAT1 mRNA was used as a control for the nuclear fraction. ( E ) HepG2 cells were treated with erastin (10 μM) for 24 h, and GABPB1-AS1 mRNA levels were assayed. ( F ) The stability of GABPB1-AS1 mRNA over time was measured after blocking new RNA synthesis with α-amanitin (50 μM) and then treating the cells with (+) or without (−) erastin. GABPB1-AS1 mRNA levels were normalized to 18 S rRNA levels. Values are expressed as the means ± SD (n = 3). *P < 0.05, **P < 0.01.

Article Snippet: ChIP was performed on the chromatin extract from HepG2 cells according to the instructions provided by Merck-Millipore.

Techniques: Expressing, Control, Blocking Assay

Journal: Scientific Reports

Article Title: LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

doi: 10.1038/s41598-019-52837-8

Figure Lengend Snippet: GABPB1-AS1 negatively regulates GABPB1 through a post-translational mechanism. ( A ) Relative expression of GABPB1-AS1 determined by RT-qPCR in HepG2 cells transfected with pcDNA3.1 or GABPB1-AS1 overexpression vector. ( B ) RT-qPCR results showing GABPB1 mRNA expression in HepG2 cells transfected with pcDNA3.1 or GABPB1-AS1 overexpression vector. ( C ) Western blot analysis of GABPB1 in HepG2 cells transfected with pcDNA3.1 or GABPB1-AS1 overexpression vector. ACTIN was used as a loading control for RNA and total protein levels. ( D ) RT-qPCR confirmed the downregulation of GABPB1-AS1 using two different siRNAs compared with the negative control (NC). ( E , F ) RT-qPCR and western blot analysis of GABPB1 mRNA levels ( E ) and protein levels ( F ) after GABPB1-AS1 expression was reduced. ( G , H ) RT-qPCR ( G ) and western blot ( H ) confirmed the downregulation of GABPB1 mRNA and protein by two different siRNAs. ( I ) RT-qPCR analysis of GABPB1-AS1 mRNA levels after GABPB1 expression was reduced. Values are expressed as the means ± SD (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: ChIP was performed on the chromatin extract from HepG2 cells according to the instructions provided by Merck-Millipore.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Negative Control

Journal: Scientific Reports

Article Title: LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

doi: 10.1038/s41598-019-52837-8

Figure Lengend Snippet: GABPB1-AS1 suppresses GABPB1 translation through direct binding with GABPB1 mRNA. ( A ) Schematic showing GABPB1-AS1 overlapping with the first exon of GABPB1. ( B ) RNase protection assay performed on RNA samples from HepG2 cells; GABPB1 mRNA regions overlapping and non-overlapping with GABPB1-AS1 were examined. ( C ) RNA pull-down assays were performed for endogenous GABPB1-AS1 mRNA using in vitro transcribed biotin-labelled GABPB1 exon 1 (sense) or antisense sequence. ( D ) Polysomes from HepG2 cells transfected with siGABPB1-AS1, GABPB1-AS1 overexpression vector or the respective controls were fractioned through sucrose gradients, and the relative distribution of GABPB1 mRNA in the gradients was measured by RT-qPCR. Data were normalized as polysome/nonpolysome. ( E ) Immunoprecipitation and western blot of eIF4A from HepG2 cells transfected with siGABPB1-AS1, GABPB1-AS1 overexpression vector or the respective controls. ( F ) RIP was performed for GABPB1 mRNA using anti-eIF4A in HepG2 cells transfected with siGABPB1-AS1, GABPB1-AS1 overexpression vector or the respective controls. Values are expressed as the means ± SD (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: ChIP was performed on the chromatin extract from HepG2 cells according to the instructions provided by Merck-Millipore.

Techniques: Binding Assay, Rnase Protection Assay, In Vitro, Sequencing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

doi: 10.1038/s41598-019-52837-8

Figure Lengend Snippet: GABPB1-AS1 regulates the erastin-induced ROS and MDA levels and abnormal expression of GABPB1 in HCC tissues. ( A ) HepG2 cells were transfected with siGABPB1-AS1, GABPB1-AS1 overexpression vector or the respective controls and then treated with erastin (10 μM) for 24 h. DCFH-DA stained cells were observed by fluorescence microscopy. Bars: 50 μm. ( B ) Western blot analysis of GABPB1 protein in HepG2 cells treated as indicated. ( C ) ROS generation in cells treated as indicated was measured by flow cytometry using DCFH-DA staining. ( D ) MDA levels were measured in cells treated as indicated. (E ) Cell viability was determined in cells treated as indicated by CCK-8 assays. ( F ) GABPB1 expression levels in normal (n = 50) and HCC primary tumour (n = 371) samples. ( G ) Kaplan-Meier analyses of the correlation between GABPB1 expression levels and overall survival. ( H ) Kaplan-Meier analyses of the correlation between GABPB1-AS1 expression levels and overall survival. ( I ) The expression levels of GABPB1-AS1 and GABPB1 were analysed using RT-qPCR in human HCC tissues (n = 12 independent samples), and the ΔCT (threshold cycle) values were normalized to GAPDH mRNA and subjected to Pearson correlation analysis. Values are expressed as the means ± SD (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: ChIP was performed on the chromatin extract from HepG2 cells according to the instructions provided by Merck-Millipore.

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Fluorescence, Microscopy, Western Blot, Flow Cytometry, CCK-8 Assay, Quantitative RT-PCR

Journal: Journal of Diabetes Research

Article Title: Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes

doi: 10.1155/2019/9582714

Figure Lengend Snippet: Triglycerides and diacylglycerol are increased by HFD. (a–d) Oil Red O staining showed a marked increase in fat droplets in mouse hepatic tissue following high fat (c) and high fat with carrageenan (d) with a little sign of fat droplets in the carrageenan-treated (b) or control hepatic tissue (a). Scale bar = 50 μ m. (e) Triglycerides were measured in the hepatic and pancreatic tissues of the study mice and increased following HFD but not carrageenan ( p < 0.001, n = 12). (f) DAG levels in the hepatic and pancreatic tissues increased following HFD in the mice but not following carrageenan exposure ( p < 0.001, n = 12). (g) Palmitic acid increased DAG in the HepG2 cells ( p < 0.001, n = 3). CGN = carrageenan; DAG = diacylglycerol; PA = palmitic acid.

Article Snippet: Diacylglycerol (DAG) concentration was determined in tissue homogenates and in HepG2 cell extracts by a competitive enzyme immunoassay, as per directions (Aviva Systems Biology, San Diego, CA).

Techniques: Staining, Control

Journal: Journal of Diabetes Research

Article Title: Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes

doi: 10.1155/2019/9582714

Figure Lengend Snippet: Serum galectin-3 increased following carrageenan or HFD. (a) Serum galectin-3 increased following carrageenan or HFD or their combination ( p < 0.001, n = 18). (b) Galectin-3 binding with the insulin receptor increased in the liver and muscle membrane preparations of the treated mice, with the greatest effect following the combined exposure ( p < 0.001, n = 12). (c) In HepG2 cells, the insulin-induced increase in phospho(Tyr)-IRS1 was significantly inhibited by administration of exogenous recombinant human galectin-3 ( p < 0.001, n = 3). (d) The insulin-induced glucose uptake in the HepG2 cells was blocked by administration of exogenous recombinant human galectin-3 ( p < 0.001, n = 3). (e) The Pearson correlation r between the glucose uptake and the phospho(Tyr)-IRS-1 in the HepG2 cells was 0.985. (f) Activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase), which removes 4-sulfate groups from the nonreducing end of chondroitin 4-sulfate and dermatan sulfate, was inhibited by exposure to carrageenan, but not by the HFD, in the liver and pancreas of the treated mice ( p < 0.001, n = 12). ARSB = arylsulfatase B; CGN = carrageenan; IRS = insulin receptor substrate.

Article Snippet: Diacylglycerol (DAG) concentration was determined in tissue homogenates and in HepG2 cell extracts by a competitive enzyme immunoassay, as per directions (Aviva Systems Biology, San Diego, CA).

Techniques: Binding Assay, Membrane, Recombinant, Activity Assay

Journal: PLoS ONE

Article Title: Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

doi: 10.1371/journal.pone.0073097

Figure Lengend Snippet: (A–C) HepG2 (A), Hep3B (B) and HuH7 (C) hepatoma tumor cells were treated with different concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control and monitored for an additional time period of 96 h. Cellular impedance was measured over the entire observation time using the xCELLigence™ SP system and calculated by the RTCA Software 1.2.1.1002. All cell index (CI) values were normalized when treatment started. Displayed are normalized CI values every 5 h. As a positive control for cell death, Triton X-100 0.1% was used. (D–F) Sulforhodamine B (SRB) assay of HepG2 (D), Hep3B (E) and HuH7 (F) cells treated with increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control for 96 h. Shown are mean ± SD of three independent experiments, each performed in triplicates (A–F); One-way ANOVA Dunnetts multiple comparison test, * P <0.01 (D–F).

Article Snippet: For HepG2 HDAC inhibition determination, nuclear extract of HepG2 cells (Active Motif) was used.

Techniques: Solvent, Control, Software, Positive Control, Sulforhodamine B Assay, Comparison

Journal: PLoS ONE

Article Title: Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

doi: 10.1371/journal.pone.0073097

Figure Lengend Snippet: (A) Detection of intracellular acetylated protein levels in HepG2, Hep3B and HuH7 hepatoma cells after incubation of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control for 6 h. (B) Overall HDAC inhibition in nuclear extracts of HepG2 cells by increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control. As a reference inhibitor suberoylanilide hydroxamic acid (SAHA; 100 µM) was used. (C) Western blot analysis of acetylated histone complex H3 in HepG2 tumor cells treated with 50 µM and 100 µM of resveratrol or solvent as control. Acetylation of H3 was examined using cellular lysates. Equal protein loading was verified by vinculin staining (upper row). As a reference and positive control for hyperacetylation the cells were treated with 2 µM SAHA. Acetylation levels were calculated performing a densitometric analysis. Shown are mean ± SD of three independent experiments, each performed in triplicates (A and B); One-way ANOVA Dunnetts multiple comparison test, * P <0.01, n.s. indicates not significant (A and B).

Article Snippet: For HepG2 HDAC inhibition determination, nuclear extract of HepG2 cells (Active Motif) was used.

Techniques: Incubation, Solvent, Control, Inhibition, Western Blot, Staining, Positive Control, Comparison