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: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 2. Hypoxia increases the expression of NR4A1 and HIF-1α and promotes glycolysis in PASMCs. (A) Cell morphology was observed by a light microscopy, scale bar = 100 μm. (B) CCK8 assays (absorbance at OD450 nm) were used to measure the cell viability of PASMCs at 0 and 24 h. (C) The levels of NR4A1 and HIF-1α were examined in PASMCs. (D) Relative mRNA levels of PKM2, GLUT1, and HK2 were examined. (E) Extracellular glucose and lactate levels were determined. n = 3. ****p < 0.0001 vs. Normoxia.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Light Microscopy

Journal: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 4. Overexpression of NR4A1 promotes glycolysis in hypoxia-induced PASMCs. (A) The effects of oe-NC and oe-NR4A1 on hypoxia- induced PASMC viability (absorbance at OD450 nm) by CCK8. (B) Relative protein expression of NR4A1 and HIF-1α determined in hypoxia- induced PASMCs. (C) The effects of NR4A1 on PKM2, GLUT1, HK2, and CD36 mRNA levels were evaluated. (D) The effects of NR4A1 on PKM2, GLUT1, HK2, and CD36 levels were measured. (E) Extracellular glucose and lactate levels were determined. n = 3. ****p < 0.0001 vs. oe-NC.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Over Expression, Expressing

Journal: BIOCELL

Article Title: NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1α expression

doi: 10.32604/biocell.2023.044459

Figure Lengend Snippet: FIGURE 5. NR4A1 induces HIF-1α expression to promote cell proliferation and glycolysis in hypoxia-exposed PASMCs. (A) Hypoxic PASMC viability (absorbance at OD450 nm) was measured by CCK8. (B) Protein levels of NR4A1 and HIF-1α were determined in hypoxia-induced PASMCs. (C) Relative mRNA levels of PKM2, GLUT1, HK2, and CD36 were examined in hypoxia-induced PASMCs. (D) Protein levels of PKM2, GLUT1, HK2, and CD36 were determined in hypoxia-induced PASMCs. (E) Determination of extracellular glucose and lactate levels in hypoxia-induced PASMCs. n = 3. ****p < 0.0001 vs. si-NC. ###p < 0.001, ####p < 0.0001 vs. si-NR4A1+oe-NC.

Article Snippet: The membranes were blocked in 5% skimmed milk for 90 min and then incubated overnight at 4°C with NR4A1 (1:600, 25851-1-AP, Proteintech, Rosemont, IL, USA), HIF-1α (1:1000, ab179483, Abcam, Cambridge, MA, UK), PKM2 (1:4000, 15822-1-AP, Proteintech), GLUT1 (1:4000, 21829-1-AP, Proteintech), HK2 (1:5000, 22029-1- AP, Proteintech), CD36 (1:1000, 18836-1-AP, Proteintech), and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Expressing

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 1. Inhibition of HIF-hydroxylase activity by CoCl2. (a) In vitro prolyl hydroxylase activity assay. The GST-HIF1a-TADN fusion protein or the GST protein was incubated with HepG2 cell extract, cofactors, and [5-14C]2-oxoglutarate in the presence of CoCl2 (10 mM). The radioactivity associated to 14C-succinate was determined. In each experi- ment, the basal HIF-TADN-dependent activity (control) was set to 100% after being normalized by subtracting the GST-associated activity. Values are means ± SEM of three independent culture experiments. Statistics, Student’s t-test for paired values: *P £ 0.05 vs. control. (b) GST pull-down assay. HepG2 cells were treated with or without CoCl2 (10 mM). Cell extracts were prepared and incubated with the GST-HIF1a-TADN fusion protein supplemented with cofactors. Glutathione-Sepharose beads and [35S]VHL were then added and the bound VHL was recovered, subjected to SDS–PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]VHL. The two bands represent the 213 and 160 amino acid VHL translation products (105, 106).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Activity Assay, In Vitro, Incubation, Radioactivity, Control, Pull Down Assay, SDS Page

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 2. Inhibition of the IGF-1-mediated HIF-1alpha induction by the PI(3)-kinase inhibitor LY294002 and the MEK inhibitor U0126. Serum-starved HepG2 cells were pretreated with 10 µM LY294002 or 10 µM U0126 for 30 min and then treated either with or without 100 nM human IGF-1 (Sigma) and exposed to normoxia (16% O2) or hypoxia (8% O2) for 4 h. Acetic acid was used in controls at a final concentration of 100 nM to keep the pH constant. 100 µg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1. Autoradiographic signals were detected by chemiluminescence (77).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Concentration Assay, Western Blot, Transduction

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: C3A viability and LC 50 values of the 40 and 80 nm a AuNP and b PC AuNP. Data represent mean ± S.D. ( n = 3). PC human plasma protein corona, ND not determined, BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, LC 50 median lethal concentration

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time-dependent cellular uptake of the 40 nm a BPEI-AuNP, b LA-AuNP, and c PEG-AuNP, and the 80 nm d BPEI-AuNP, e LA-AuNP, and f PEG-AuNP in the absence and presence of PC in C3A cells up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona, MSD minimum significant difference. * p < 0.05; ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time- and concentration-dependent ROS/RNS production in C3A cells exposed to a the 40 nm BPEI-AuNP and b the 40 nm PEG-AuNP up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, CTRL control, MSD a minimum significant difference, PCN pyocyanin (ROS inducer). ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Concentration Assay, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: An inhibitory effect of AuNP on CYP3A4 activity in C3A cells exposed to the 40 and 80 nm BPEI-, LA-, and PEG-AuNP in the absence and presence of PC for 24 h. Values represent mean ± S.D. ( n = 3). BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Activity Assay, Clinical Proteomics

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: Endocrinology

Article Title: Endogenous IGFBP-3 Mediates Intrinsic Apoptosis Through Modulation of Nur77 Phosphorylation and Nuclear Export.

doi: 10.1210/en.2015-1215

Figure Lengend Snippet: Figure 1. Endogenous Nur77 is knocked down using two different siRNA oligos and is detectable in MAC-T cells with a commercially available antibody. Cells were transfected with scramble (SCR) or Nur77 siRNA (Oligo No. 1 or Oligo No. 2). Total RNA (A) or protein (B) was isolated from WCLs after 48 h and analyzed for Nur77 by RT-qPCR (corrected for cyclophilin) or Western immunoblot (50 g protein), respectively. For the immunoblot, HSP60 served as a loading control. Bars represent mean SE of three experiments. C, To confirm specificity of the Nur77 antibody, MAC-T WCL (50 g), WCL from MAC-T cells transfected with myc-tagged Nur77 (3 g), HepG2 WCL (50 g), and nuclear fractions from MAC-T cells (5 g) were immunoblotted for Nur77 on one half of the membrane and myc on the other half. Actin served as a loading control.

Article Snippet: HepG2 cell lysate was obtained from Novus Biologicals.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Western Blot, Control, Membrane

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on hepatic apoC-III expression. Rat primary hepatocytes were transduced with Foxo1 or LacZ vector at an MOI of 50 PFU/cell or mock-transduced with PBS. After 24 hours of transduction, the intracellular levels of apoC-III (A), Foxo1 (B), and GK (C) mRNA were determined by real-time RT-PCR using β-actin mRNA as control. The effect of Foxo1 on hepatic apoC-III expression in response to insulin was assayed in HepG2 cells. Cells were transduced with Foxo1, Foxo1-ADA, or control LacZ vector (50 PFU/cell) in the absence or presence of insulin at different concentrations. Twenty-four hours after transduction, cells were collected for determination of the intracellular levels of apoC-III mRNA induced by Foxo1 (D) and Foxo1-ADA (E). *P < 0.05, **P < 0.005; significantly different from controls. NS, not significant by ANOVA. Data were from 3 independent experiments.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Expressing, Transduction, Plasmid Preparation, Quantitative RT-PCR

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on the human APOC3 promoter activity. (A) The APOC-III promoter–directed luciferase reporter system. The wild-type and mutant IRE sequences are underlined. (B) Foxo1-mediated induction of the APOC3 promoter activity. HepG2 cells were transfected by pHD317 together with Foxo1 construct, or with both Foxo1 and Foxo1-Ø256 constructs. For each construct, 1 μg of DNA for each construct was used in transfection. For normalization of transfection efficiency, 1 μg pCMV5-LacZ DNA was included for normalization of transfection efficiency. (C) The APOC3 promoter variants in the luciferase reporter system. (D) Responses of APOC3 promoter variants to Foxo1 production. HepG2 cells were transfected with individual test plasmids in the absence (–) or presence (+) of pCMV5-Foxo1. The relative luciferase activity, after normalizing to β-gal activity, was compared between basal (–) and Foxo1-inducible (+) conditions. (E) Responses of wild-type and mutant APOC3 promoters to insulin. Test plasmids were transduced into HepG2 cells in the presence and absence of pCMV5-Foxo1 transfection in culture media, either supplemented with or without insulin (30 nM). The relative luciferase activity in transduced cells was determined using β-gal activity as control. *P < 0.001 vs. controls.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Activity Assay, Luciferase, Mutagenesis, Transfection, Construct

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Molecular interaction between Foxo1 and the APOC3 promoter. Molecular association between Foxo1 and the APOC3 promoter was analyzed by EMSA and ChIP. Aliquots of Foxo1 protein from linked in vitro transcription-translation products (5 μg) were incubated with 2.5 μl of radioactively labeled DNA corresponding to –467/–440 nt in the human APOC3 promoter (WT-IRE) (A), a mutant APOC3 IRE (mt-IRE) containing 2 substitutions, of –A458C and –A460G, and a control PEPCK IRE DNA (B), followed by electrophoresis through 8% nondenaturing polyacrylamide gels for 30 minutes. Lane 1, DNA probe alone. Lane 2, DNA probe + Foxo1 protein lysates. Lane 3, DNA probe + Foxo1 protein lysates + anti-Foxo1 antibody (1 μg). Lane 4, DNA probe + Foxo1 protein lysates + nonlabeled competitor DNA at a molar concentration of 50-fold excess. Free, shifted, and supershifted DNA bands were visualized by autoradiography. For ChIP assay, HepG2 cells were transduced with Foxo1 vector at an MOI of 50 PFU/cell. Cells were harvested 24 hours later and subjected to ChIP using PBS as a negative control (lane 5), control IgG (lane 6), and anti-Foxo1 antibody (lane 7). The coimmunoprecipitated chromatin DNA was analyzed by immunoblot (C) using anti-Foxo1 antibody and PCR (D) using the primers that correspond to –655/–20 nt of the APOC3 promoter.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: In Vitro, Incubation, Labeling, Mutagenesis, Electrophoresis, Concentration Assay, Autoradiography, Transduction, Plasmid Preparation, Negative Control, Western Blot