HepG2/C3 cell line gene expression profiling in response to HBCD and DMBA
Ontology highlight
ABSTRACT: SurePrint G3 Human Gene Expression 8x60k oligonucleotide microarrays (Design ID: 039494,Agilent) were used to characterize gene expression profiles of HepG2/C3A cells exposed to HBCD (hexabromocyclododecane) or DMBA (dimethylbenzanthracene) for 24 hours.
Project description:SurePrint G3 Human Gene Expression 8x60k oligonucleotide microarrays (Design ID: 028004,Agilent) were used to characterize gene expression profiles of A549 cells exposed to different concentrations of HBCD (hexabromocyclododecane) for 24 hours.
Project description:Human HepG2/C3A cells were exposed to indoor dust reference material SRM2585; DMBA (dimethylbenzanthracene); HBCD (hexabromocyclododecane); two different mixtures of flame retardants (all dissolved in 0.1% DMSO) or 0.1% DMSO alone for 72h. RNA was prepared and labeled with Cy3 then hybridized to Agilent SurePrint G3 Human GE v2 8x60k Microarrays, Agilent design ID 039494.
Project description:Human cell cultures A549 and HepG2/C3 were exposed to 0-100 micromolar Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) in 0.5% DMSO or to 0.5% DMSO alone for 24 h. RNA was prepared and labeled with Cy3. Agilent SurePrint G3 Human GE v2 8x60k Microarrays, Agilent design ID 039494, were used to profile transcriptional responses to TDCIPP.
Project description:Gene expression profiles comparing FCCP, CCCP, Dinoterb, Benzoapyrene, TCP, Medium control, 0,1% DMSO in C3A cells C3A cells were exposed for 2 hours to substances, 6 replicate wells per concentration were pooled. Total RNA was extracted using RNeasy Mini Kit (QUIAGEN, Product 74104, Hombrechtikon, Switzerland). Quality of extracted RNA was checked via the Nanodrop 1000 spectraphotomoeter V3.7, as well as running the samples on a 1.5% agarose gel. cDNA was generated and labeled with Cy3 or Cy5-CTP using the low RNA input linear amplification kit (Perkin-Elmer). Total RNA was reverse transcribed to cDNA using RNasin® Plus RNAse Inhibitor (N2611), M-MLV Reverse transcriptase (RNase H Minus, Point Mutant) (M3682), 5x Buffer (M3682) and random primer (C1181) from Promega AG (product number in brackets) (Promega AG, Dübendorf, Switzerland). For Microarray analysis Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used. Each treatment consisted of two biological replicates, the control consisted of three biological replicates.
Project description:Gene expression profiles comparing FCCP, CCCP, 6OH-BDE47, Ethylenglycol, TCP, PCP, Aniline, chloroaniline, DMSO in C3A cells C3A cells were exposed for 2 hours to substances, 6 replicate wells per concentration were pooled. Total RNA was extracted using RNeasy Mini Kit (QUIAGEN, Product 74104, Hombrechtikon, Switzerland). Quality of extracted RNA was checked via the Nanodrop 1000 spectraphotomoeter V3.7, as well as running the samples on a 1.5% agarose gel. cDNA was generated and labeled with Cy3 or Cy5-CTP using the low RNA input linear amplification kit (Perkin-Elmer). Total RNA was reverse transcribed to cDNA using RNasin® Plus RNAse Inhibitor (N2611), M-MLV Reverse transcriptase (RNase H Minus, Point Mutant) (M3682), 5x Buffer (M3682) and random primer (C1181) from Promega AG (product number in brackets) (Promega AG, Dübendorf, Switzerland). For Microarray analysis Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used. Each treatment and controls consisted of three biological replicates.
Project description:The results of this study demonstrated that esculetin can affect the glucose metabolism by binding to glycolytic proteins, thus playing an anti-tumor role, and the pathway comprising these proteins which have direct interactions are a potential novel targets for tumor treatment by esculetin.
Project description:Glycolysis can improve the tolerance of tissue cells to hypoxia, and its intermediates provide raw materials for the synthesis and metabolism of the tumor cells. If it can inhibit the activity of glycolysis-related enzymes and control the energy metabolism of tumor, it can be targeted for the treatment of malignant tumor. The target proteins phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2) and glucose-6-phosphate isomerase (GPI) were screened by combining transcriptome, proteomics and reverse docking. We detected the binding constant of the active compound using microscale thermophoresis (MST). It was found that esculetin bound well with three potential target proteins. Esculetin significantly inhibited the rate of glycolysis, manifested by differences of cellular lactate production and glucose consumption in HepG2 cells with or without esculetin. It was found that GPD2 bound strongly to GPI, revealing the direct interaction between the two glycolysis-related proteins. Animal tests have further demonstrated that esculetin may have anticancer effects by affecting the activity of PGK2, GPD2 and GPI. The results of this study demonstrated that esculetin can affect the glucose metabolism by binding to glycolytic proteins, thus playing an anti-tumor role, and these proteins which have direct interactions are potential novel targets for tumor treatment by esculetin.
Project description:The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, toxicogenomics predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we aimed to determine whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test sites adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen B[a]P and the human hepatocyte cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were still observed between the test sites, a common subset of B[a]P responsive genes was identified across all test sites which included both genes previously reported in the literature as B[a]P responsive in addition to the same most highly up and down regulated genes. Pending further analysis, these preliminary data show promise that the current generation of microarray technology in combination with a standard in vitro experimental design can produce robust data that can be reproducibly generated in independent laboratories. Future work will need to determine whether in vitro model(s) can be not only reproducible but also predictive for a range of toxic chemicals with different mechanisms of action. Such an approach may potentially be part of future in vitro testing regimes for regulatory risk assessment. The aim of this study was to evaluate the inter- and intra-laboratory reproducibility of toxicogenomics data for toxicity testing in a regulatory setting. 3 Test Centres performed the same experimental protocol treating HepG2 cells with 3 mM Benzo[a]pyrene (B[a]P) (in 0.5% DMSO) for 24 hours prior to RNA extraction and microarray analysis. Three biological replicates of B[a]P and DMSO treatments were analysed by microarray analysis in each Test Centre. Two Test Centres analysed one sample in triplicate to determine technical reproducibility of the chosen array platform. One Centre analysed two samples in duplicate. 24 samples in total were used in this study
Project description:The application of RNA-seq profiling of cell lines following compound treatment has been highlighted as a line of evidence for Next Generation Risk Assessment of ingredients. We demonstrate as part of a use case the evaluation of a targeted RNAseq using Tempo-Seq platform as a general unbiased approach to complement more targeted approaches. We show impact of the use of different cell lines to the point of departure.
Project description:We have grown hepatocyte spheroids for 21 days following an established protocol. We have carried out proteomic analysis of the difference between control and treated (Fatty acids) spheroids during seven days (5 timepoints at day 0, 1, 2, 4 and 7). Samples were digesting using filter aided sample preparation-based digestion and subsequently analysed using label free LC-MS/MS.