Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: Titration of AAV2/8 Luc in HepG2 and Huh-7 liver cancer cells. Three multiplicities of infection (MOI) doses (10 4 , 10 5 , 10 6 —viral genomes) were tested in HepG2 ( upper panel) and in Huh-7 liver cancer cells ( lower panel) at three time points (24 h, 48 h, 72 h). Untransduced cells (CTL) were used as negative controls. Data are presented as mean ± SEM, n = 3.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Titration, Infection

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: mRNA expression of SIRT6 WT or SIRT6 Cent in HepG2 ( A ) and in Huh-7 ( B ) liver cancer cells. Cells were transduced with AAV2/8-Luc, AAV2/8-SIRT6-WT and AAV2/8-SIRT6-N308K/A313S for 48 h. mRNA levels of SIRT6 WT and SIRT6c were assessed by qPCR. Data are presented as mean ± SEM, n = 3. p < 0.001 (***) versus AAV2/8-Luc and AAV2/8-SIRT6-N308K/A313S-transduced cells.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Expressing, Transduction

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: MTT viability assays: MTT assay on HepG2 ( A ); and Huh-7 liver cancer cell line ( B ) transduced with AAV2/8-Luc, AAV2/8-SIRT6-WT and AAV2/8-SIRT6-N308K/A313S for up to five days (0 h, 24 h, 48 h, 72 h, 96 h, 120 h). Cell survivals were significantly lower in AAV2/8-SIRT6-WT and AAV2/8-SIRT6-N308K/A313S-transduced cells from AAV2-8-Luc at the indicated time points, with a p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). Data are presented as mean ± SEM, n = 3.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: MTT Assay, Transduction

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: Global transcriptomic profiling in hepatocellular carcinoma cells: two-dimensional principal component analysis (PCA) plots derived from variance-stabilized expression values (DESeq2) for HepG2 ( A ); and Huh7 ( B ) liver cancer cells. Each point represents an individual biological replicate, illustrating clear separation of samples according to experimental condition (Luc control, SIRT6-WT, and SIRT6-Cent), reflecting distinct overall gene-expression signatures.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Derivative Assay, Expressing, Control, Gene Expression

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: Differential expression and pathway analysis in HepG2 liver cancer cells: ( A ) volcano plots displaying differentially expressed genes across the three pairwise comparisons (SIRT6-WT vs. Luc, SIRT6-Cent vs. Luc, and SIRT6-Cent vs. SIRT6-WT). The horizontal axis shows log 2 fold-change, and the vertical axis indicates −log 10 (adjusted p -value). Upregulated genes are shown in blue, downregulated genes in red; and ( B ) reactome pathway enrichment results for the same comparisons. The bubble size corresponds to the number of differentially expressed genes contributing to each pathway, while color intensity reflects the degree of statistical enrichment.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Quantitative Proteomics

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: Invasive capacity of HCC cells after SIRT6 overexpression: invasion through basement membrane-coated inserts was evaluated in HepG2 ( A ); and Huh7 ( B ) liver cancer cells 48 h after transduction with AAV2/8-Luc (control), AAV2/8-SIRT6-WT, or AAV2/8-SIRT6-N308K/A313S, followed by a 24 h migration period toward 10% FBS as chemoattractant. White bars: no chemoattractant (negative control); black bars: migration inducer provided with the kit (positive control). Both SIRT6-WT and the centenarian-associated SIRT6 variant dramatically reduced cell invasion compared with the Luc control. Data are presented as mean ± SEM, n = 3. Data are presented as mean ± SEM, n = 3. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 versus AAV2/8-Luc; $$$ p < 0.001 versus AAV2/8-SIRT6-WT.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Over Expression, Membrane, Transduction, Control, Migration, Negative Control, Positive Control, Variant Assay

Journal: Cancers

Article Title: Transcriptional Control of Hepatocellular Carcinoma Cells Aggressiveness by AAV2/8-Mediated Delivery of Human Centenarian-Associated SIRT6 N308K/A313S

doi: 10.3390/cancers18050812

Figure Lengend Snippet: Nanoindentation assays in: HepG2 ( A ); and Huh-7 liver cancer cell lines ( B ) transduced with AAV2/8-Luc, AAV2/8-SIRT6-WT and AAV2/8-SIRT6-N308K/A313S for two days prior to incubation in the Boyden chamber for a further 24 h; 10% FBS was used as chemoattractant. Cell stiffness (measured in Pascal, Pa) was significantly higher in AAV2/8- AAV2/8-SIRT6-N308K/A313S-transduced cells compared to AAV2/8-Luc and to AAV2/8-SIRT6-WT. p < 0.05 (*), p < 0.01 (**) versus AAV2/8-SIRT6-WT.

Article Snippet: HepG2 and Huh-7 liver cancer cells were obtained from CLS-GmbH (Eppelheim, Germany) and were authenticated using STR profiling.

Techniques: Transduction, Incubation

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: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Comparison of AOP1 EC 50 s evaluated in Caco-2 and HepG2 cells for the 12 natural extracts selected for the intestinal barrier transport study. Extracts were selected according to the best EC 50 s obtained with AOP1 assay. In order to increase the diversity of tested extracts, some Vitis vinifera extracts were removed from the list because of redundancy with each other. ND = not determined (no full effect observed).

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Indirect antioxidant ARE/Nrf2 assay data obtained for Syzygium aromaticum extract (given as an example) in HepG2/Nrf2 cells. All other extracts were subjected to the same protocol and data analyses. ( A ) Assay of increasing concentrations showing the typical bell-shaped profile (see text for explanation). Sulforaphane 25 µM was used as positive control; ( B ) dose–response and sigmoid fit curve used for Efficacy Concentration (EC) estimations.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Positive Control, Concentration Assay

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: ARE/Nrf2 EC 50 s established in HepG2 cells for the 12 natural extracts selected for the Caco-2 intestinal barrier transport study. ND = not determined; R 2 = determination coefficient.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of post-intestinal barrier transport compartments on ARE/NRF2 pathway activation in HepG2 cells. Results are expressed as fold increase compared to constitutive medium ARE/Nrf2 gene expression. Values represent means of quadruplicates obtained from two independent intestinal barrier transport experiments. Sulforaphane 25 µM data is provided as positive control; *** p < 0.005.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Activation Assay, Expressing, Positive Control

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of combinations of post-intestinal barrier compartments on ARE/NRF2 pathway in HepG2 cells. Results are expressed as ARE/Nrf2 gene expression fold increase compared to constitutive medium. Values represent means of pentaplicates for all 50/50 combinations and of triplicates for all others. All data were normalized to constitutive medium values; *** p < 0.001; ** p < 0.01.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Synergistic effects on ARE/Nrf2 gene expression of post-intestinal barrier compartment combinations. Combin. FI = gene expression fold increase (FI) measure for the combination; S indep. FI = sum of the increase in gene expression fold increase (FI) measures of each of the two extracts taken independently as depicted in Figure 6 ; Var. (%) = variation in percentage of the two values. A positive percentage value indicates a synergistic effect.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Glucose-coated superparamagnetic iron oxide nanoparticles prepared by metal vapor synthesis can target GLUT1 overexpressing tumors: In vitro tests and in vivo preliminary assessment

doi: 10.1371/journal.pone.0269603

Figure Lengend Snippet: GLUT1 levels estimated by ELISA in PSN-1, BCPAP and HEK293 cells (at the top). Uptake studies: PSN-1 and BCPAP cells were incubated with 0.1 mg/mL of Glc-SPIONs for 30 min, 1, 3, 6, and 12 h. The Fe cellular content was estimated by GF-AAS analysis. Uptake studies after treating the cells with GLUT1 inhibitors: PSN1 and BCPAP cells were pre-incubated for 1 h with anti-GLUT1 polyclonal antibody, WZB117, Fasentin, BAY-876, and STF-31. Subsequently, cells were treated for 3 or 6 h with 0.1 mg/mL of Glc-SPIONs.

Article Snippet: Expression levels in cancer cells were evaluated by means of the GLUT1 Colorimetric Cell-Based ELISA kit (Boster Biological Technology, Pleasanton CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: PLoS ONE

Article Title: Construction of the experimental rat model of gestational diabetes

doi: 10.1371/journal.pone.0273703

Figure Lengend Snippet: (A) Effects on the protein expression levels of GLUT1 and GLUT3 and the internal reference β-actin as determined by Western blot analysis, (B) Comparison of the protein expression levels of GLUT1 and GLUT3. * P < 0.05 vs. the NC group; # P < 0.05 vs. the HFHS group.

Article Snippet: After blocking with 5% skimmed milk in PBS-Tween, the membranes were probed with anti-glucose transporter 1 (GLUT1) or anti-glucose transporter 1 (GLUT3) primary antibody (1:500, Boeter) at 4°C overnight and conjugated with secondary antibodies at room temperature for 45 min. Antibodies against GLUT1 and GLUT3 were obtained from BOSTER Biological Technology Co., Ltd.

Techniques: Expressing, Western Blot, Comparison

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