Journal: Pathogens

Article Title: A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

doi: 10.3390/pathogens11030355

Figure Lengend Snippet: Luciferase activity of four parental HEV replicons in comparison to mock-transfected control (H 2 O) and inactivated RdRp GAA mutant replicon based on p6LucA26. HepG2 cells were transfected with replicon ivtRNA by electroporation and seeded across four wells of a 96-well plate as technical quadruplicate. Replication was estimated by measuring total luciferase activity (relative light units; RLU) of each replicon. Error bars indicate standard deviation and asterisks indicate significance level (*** p ≤ 0.001; ** 0.001 < p ≤ 0.01; ns 0.05 < p ≤ 1). The data depict a representative experiment of three separate transfections.

Article Snippet: HepG2 cells were purchased from CLS (Eppelheim, Germany) and grown in DMEM (Gibco 52100, ThermoFisher, Waltham, MA, USA), supplemented with NaHCO 3 and Na-pyruvate, and 10% fetal bovine serum, including 10 mg/L gentamicin (PAN-Biotech, Aidenbach, Germany), and 250 mg/L amphotericin B (PAN-Biotech, Aidenbach, Germany) in cell culture flasks with vented caps (Corning, Corning, NY, USA) in a humidified incubator with 5% CO 2 .

Techniques: Luciferase, Activity Assay, Comparison, Transfection, Control, Mutagenesis, Electroporation, Standard Deviation

Journal: Pathogens

Article Title: A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

doi: 10.3390/pathogens11030355

Figure Lengend Snippet: ( A ) Structure of nine chimeric replicons that were constructed using p6LucA26 as backbone. Subgenomic fragments containing partial sequences of ORF1 (MYP, nucleotide positions 1–2139 in p6; VXH, nucleotide positions 2144–4059 in p6; RJ, nucleotide positions 4064–5347 in p6) were replaced with the corresponding sequences from HEV strains 47832mc, rab52, and rab81. Percentages annotated in the highlighted fragments represent pairwise amino acid sequence identity of the encoded proteins with the corresponding ones encoded by parental p6 replicon. Multiple amino acid sequence alignments of each ORF1-encoded protein segment are included in the . ( B ) HepG2 cells were transfected with replicon ivtRNA by electroporation and seeded across four wells of a 96-well plate as technical quadruplicates. Replication was estimated by measuring total luciferase activity (RLU) of each replicon. Error bars indicate standard deviation; asterisks indicate significance level in reference to p6LucA26 (**** p ≤ 0.0001; *** 0.0001 < p ≤ 0.001; ** 0.001 < p ≤ 0.01; * 0.01 < p ≤ 0.05; ns 0.05 < p ≤ 1). The data depict a representative experiment of three separate transfections. Note that the controls (p6LucA26, p6GAALucA26, and mock) are shown in each plot for illustrative purposes. The data depicted are derived from a single experiment with all chimeric replicons and one group of controls.

Article Snippet: HepG2 cells were purchased from CLS (Eppelheim, Germany) and grown in DMEM (Gibco 52100, ThermoFisher, Waltham, MA, USA), supplemented with NaHCO 3 and Na-pyruvate, and 10% fetal bovine serum, including 10 mg/L gentamicin (PAN-Biotech, Aidenbach, Germany), and 250 mg/L amphotericin B (PAN-Biotech, Aidenbach, Germany) in cell culture flasks with vented caps (Corning, Corning, NY, USA) in a humidified incubator with 5% CO 2 .

Techniques: Construct, Sequencing, Transfection, Electroporation, Luciferase, Activity Assay, Standard Deviation, Derivative Assay

Journal: Scientific Reports

Article Title: Insulin signaling and glucose metabolism in different hepatoma cell lines deviate from hepatocyte physiology toward a convergent aberrant phenotype

doi: 10.1038/s41598-020-68721-9

Figure Lengend Snippet: The HepG2 human hepatoma cell line displays aberrant insulin signaling compared to murine primary hepatocytes. ( a ) Immunoblot analysis of insulin signaling in primary mouse hepatocytes infected with a control adenovirus expressing the GFP or a virus expressing the negative dominant RAS mutant H-RAS17N and stimulated with 10 nM insulin for 8 min. ( b ) Quantification of blots in ( a ). ( c ) Immunoblot analysis of insulin signaling and insulin receptor phosphorylation performed as in ( a ) but using the HepG2 cell line. ( d ) Quantification of blots in ( c ). n = 3 biological replicates (mice) for primary hepatocytes and 3 independent experiments for HepG2. Data are represented as mean ± SEM.

Article Snippet: Thereby, we have examined the information available on the HepG2 cell line at Broad Institute Cancer Cell Line encyclopedia (CCLE), where we have found 386 entries for mutations in the HepG2 genome.

Techniques: Western Blot, Infection, Control, Expressing, Virus, Mutagenesis, Phospho-proteomics

Journal: Scientific Reports

Article Title: Insulin signaling and glucose metabolism in different hepatoma cell lines deviate from hepatocyte physiology toward a convergent aberrant phenotype

doi: 10.1038/s41598-020-68721-9

Figure Lengend Snippet: Hepatoma cell lines display severely impaired gluconeogenic gene expression and blunted glucose production. ( a ) qPCR analysis of mRNA levels of glucose 6 phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) in primary mouse hepatocytes exposed for 6 h to 100 µM of dbcAMP, or 10 nM insulin, or 100 µM dbcAMP and 10 nM insulin. ( b ) qPCR analysis of mRNA levels of G6P and PEPCK in Hepa 1–6 cells treated as in ( a ). ( c ) qPCR analysis of mRNA levels of G6P and PEPCK in McARH7777 cell line treated as in ( a ). ( s ) qPCR analysis of mRNA levels of G6P and PEPCK in HepG2 cells treated as in ( a ). ( e ) Direct comparison of basal and cAMP-induced G6P and PEPCK mRNA levels in primary mouse hepatocytes and mouse Hepa 1–6. ( f ) Immunoblot analysis of insulin-driven AKT Thr 308 phosphorylation, AKT Ser 473 phosphorylation and GSK3β phosphorylation in primary hepatocytes and Hepa 1–6 cells stimulated for 8 min with 10 nM insulin. ( g ) Quantification of blots in ( f ). ( h ) Relative glucose production in primary hepatocytes, Hepa 1–6, McARH7777, and HepG2 stimulated with 100 µM dbcAMP in presence or not of 10 nM insulin for 6 h. n = 8 for ( a ); n = 5 for ( b ); n = 8 for ( c ); n = 7 for ( d ); and ( e ) n = 8 for heaptocytes and n = 5 for Hepa 1–6 n = 3 for ( f , g ) n = 3; for ( h ) n = 5 hepatocytes and n = 4 for each hepatoma cell line. n indicates biological replicates for primary hepatocytes, and independent experiments for cell lines. Data are represented as mean ± SEM.

Article Snippet: Thereby, we have examined the information available on the HepG2 cell line at Broad Institute Cancer Cell Line encyclopedia (CCLE), where we have found 386 entries for mutations in the HepG2 genome.

Techniques: Gene Expression, Comparison, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: Insulin signaling and glucose metabolism in different hepatoma cell lines deviate from hepatocyte physiology toward a convergent aberrant phenotype

doi: 10.1038/s41598-020-68721-9

Figure Lengend Snippet: Hepatoma cell lines display reduced fatty acid synthase expression and a distinct protein electrophoresis pattern. ( a ) Immunoblot analysis of FAS on primary hepatocytes, Hepa 1–6, McARH7777, and HepG2. ( b ) Quantification of blots in ( a ). ( c ) Coomassie staining of a protein electrophoresis of extracts from human primary hepatocytes, murine primary hepatocytes, Hepa 1–6, McARH7777, and HepG2. n = 9 for a and b; n = 2–3 for c. n indicates biological replicates for primary hepatocytes, technical replicates for human hepatocytes, and independent experiments for cell lines. Data are represented as mean ± SEM.

Article Snippet: Thereby, we have examined the information available on the HepG2 cell line at Broad Institute Cancer Cell Line encyclopedia (CCLE), where we have found 386 entries for mutations in the HepG2 genome.

Techniques: Expressing, Protein Electrophoresis, Western Blot, Staining