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: 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

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: Effect of PB123 treatment on HepG2 cells. (A) PB123 was not toxic to HepG2 cells, measured by treating the cells for 24h with PB123 then determining cell viability by CCK8 assay. (B) PB123 activated Nrf2 in HepG2-ARE cells in a dose-dependent manner (p<0.05) by 5 and 12 μg/mL PB123.

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques: CCK-8 Assay

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: The Nrf2-inhibitor AEM1 dose-dependently blocked Nrf2 activation (p<0.05) induced in HepG2-ARE cells by treatment with PB123 (10 μg/mL).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques: Activation Assay

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: Combinatorial synergy analysis of HepG2 cells treated with Rosemary and Ginger extracts. Nrf2 activation by checkerboard combinations of Rosemary and Ginger extracts showed a strongly synergistic response as calculated using both (A) the Zero Interaction Potency and (B) the Loewe additive effect reference synergy models ( synergyfinder.org ).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques: Activation Assay

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: BioJupies was used to generate a Volcano plot of differentially expressed genes by HepG2 cells treated with PB123 12 μg/ml compared with untreated control HepG2 cells. Red indicates upregulated genes and blue indicates downregulated genes. Genes were selected with 2-fold change threshold. Gene symbols on the plots are used to mark some of the most significant genes changed, showing lipid and cholesterol synthesis genes downregulated and Nrf2-dependent genes upregulated.

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques:

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: Cholesterol Biosynthesis Pathway genes from WikiPathways (WP197, https://www.wikipathways.org/index.php/Pathway:WP197 ). In our mRNA-seq dataset we found that all of the genes were significantly downregulated (shown in blue) by 12 μg/mL PB123 in HepG2 cells except PMVK (shown in red) compared to control-treated HepG2 cells (p<0.05, n = 4 per group). HMG-CoA reductase ( HMGCR ) is the rate-limiting enzyme in the pathway.

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques:

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: HepG2 total cholesterol. Total cholesterol levels were significantly reduced in HepG2 cells by 24 treatment with 5 or 12 μg/mL PB123 compared to untreated control HepG2 cells (p<0.05, n = 12 per group).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques:

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: HepG2 intracellular lipids. We found that (A) FABP1 was significantly downregulated by both 5 and 12 μg/mL PB123 in HepG2 cells compared to untreated control HepG2 cells (p<0.05, n = 4 per group), and (B) that intracellular lipid content was likewise significantly reduced by both 5 and 12 μg/mL PB123 in HepG2 cells compared to untreated control HepG2 cells (p<0.05, n = 12 per group).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques:

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: Heme oxygenase-1 gene expression and protein were increased in HepG2 cells by PB123 treatment. Treatment of HepG2 cells with PB123 (5 μg/mL, 24h) increased the both the levels of HMOX1 gene expression determined by mRNA-seq (A) and the levels of HMOX1 protein determined by ELISA (B), as expected based on the large PB123-induced increase in Nrf2 activation (p<0.05, n = 4 in each case).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: OBM integrative and complimentary medicine

Article Title: Effects of the Phytochemical Combination PB123 on Nrf2 Activation, Gene Expression, and the Cholesterol Pathway in HepG2 Cells

doi: 10.21926/obm.icm.2201002

Figure Lengend Snippet: PB123 prevented loss of cell viability following challenge with an oxidative stress. Cytotoxicity was not observed (cell proliferation measured by CCK8 assay) in HepG2 cells treated for 16h with 5 μg/mL PB123 compared to untreated control cells. In HepG2 cells treated with 5 μg/mL PB123 or vehicle control for 18h, and then challenged with 25 μM cumene hydroperoxide (CH) or untreated control for 6 h, loss of cell viability (toxicity) was caused by CH challenge but this toxicity was partially attenuated ( p < 0.05) by PB123 pretreatment. The protective effect of PB123 was blocked (p<0.05) by ERK1/2 kinase inhibition (10 μM PD98059, 30 min prior to PB123 treatment).

Article Snippet: The Human HMOX1 PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA) was used to determine heme oxygenase-1 (HMOX1) protein levels in HepG2 cell lysates according to the manufacturer’s instructions as previously described [ ].

Techniques: CCK-8 Assay, Inhibition

Journal: Biochemical Journal

Article Title: The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

doi: 10.1042/BCJ20190647

Figure Lengend Snippet: ( A ) CHO-LDM-V5 cells were treated with 10 µg/ml cycloheximide (CHX), 20 µg/ml cholesterol/CD (Chol) or 1 µg/ml 25-hydroxycholesterol (25HC) for 8 h. ( B ) CHO-LDM-V5 cells were treated in 5% (v/v) LPDS or NBS based media with or without statin (5 µM compactin and 50 µM mevalonate) for 16 h. ( C ) HeLaT and HepG2 cells were exposed to 21% (normoxic) or 2% (hypoxic) oxygen conditions for 4 h; the lysates were obtained from Novus Biologicals. ( D ) CHO-LDM-V5 cells were treated with or without 500 µM DPTA NONOate (DPTA) for the indicated time. Protein levels were analysed by Western blotting with V5, endogenous LDM and vinculin antibodies. Except in ( C ), data are presented as mean ± SEM from at least three independent experiments ( A n = 3, B n = 5, D n = 4), where ** P < 0.01. Relative protein levels were measured using ImageStudio Lite (version 5.2) and normalised to the vehicle (Veh) or control condition in each blot, which was set to 1 ( A–C ) or 100% at each time point ( D ).

Article Snippet: Normoxic and hypoxic lysates from HeLaT and HepG2 cells were obtained from Novus Biologicals.

Techniques: Western Blot, Control

Journal: Biochemical Journal

Article Title: The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

doi: 10.1042/BCJ20190647

Figure Lengend Snippet: ( A ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM of the indicated siRNA (M6: MARCH6), then mRNA levels were measured using qRT-PCR and normalised to the housekeeping gene PBGD . mRNA levels were normalised to the control condition which was set to 1. Data are presented as mean + half range from an experiment performed in triplicate. ( B ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM of the indicated siRNA (M6: MARCH6). ( C ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM control or MARCH6 siRNA, then treated with or without 500 µM DPTA NONOate (DPTA) for 2 h. ( D ) HepG2 cells were transfected for 24 h with 25 nM control or MARCH6 siRNA, then treated with or without 10 µg/ml cycloheximide (CHX) for 8 h. ( E ) CHO-LSS-myc, CHO-LDM-V5, CHO-EBP-V5 or CHO-DHCR24-V5 cells were transfected for 24 h with 25 nM control or MARCH6 (M6) siRNA. Bands for the ectopic cholesterol synthesis enzymes migrated close to the expected size; LSS-myc: 83 kDa, LDM-V5: 57 kDa, EBP-V5: 26 kDa, DHCR24-V5: 60 kDa. Protein levels were analysed by Western blotting with V5, myc, endogenous SM, endogenous LDM and vinculin antibodies. Data are presented as mean + SEM from at least three independent experiments ( B n = 3–7, C n = 6, D n = 3, E n = 3–6), where * P < 0.05 and ** P < 0.01. Relative protein levels were measured using ImageStudio Lite (version 5.2) and normalised to the control condition which was set to 1.

Article Snippet: Normoxic and hypoxic lysates from HeLaT and HepG2 cells were obtained from Novus Biologicals.

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