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: 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: 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