Relative gene expression was evaluated using 3 reference genes: and . had an additive effect on reducing the viability and proliferation of Dt81Hepa1-6 cells. This metabolic-induced tumorigenicity was also noticed in human tumorigenic HCC cells, where we not only found that increased glucose uptake capacity of Huh7 was intrinsically associated with their degree of tumorigenicity but also that increased expression of glycolytic genes ((by tumor cells from HCC patients correlated with poor survival. Therefore, the tumorigenicity of HCC cells can stem from their ability to metabolically adapt to a nutrient-poor microenvironment. Given that increased expression of glycolytic enzymes also correlates with poor prognosis in HCC patients, new drugs that target these metabolic enzymes could be used to improve or potentiate current treatment regimen. Materials and methods Reagents Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/Streptomycin, fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2- deoxyglucose (2-NBDG), TRIZOL? Trolox reagent, MitoTracker? Red CMXRos and MitoTracker? Red CM-H2XRos were purchased from Invitrogen (Burlington, On, Canada). QuantiTect reverse transcription kit and QuantiTect SYBR Green PCR Kit were purchased Trolox from QIAGEN (Toronto, On, Canada). Unless stated otherwise, all other products were from Sigma-Aldrich (Oakville, On, Canada). Cell lines and culture conditions Authenticated Hepa1-6 murine, Huh7 and HepG2 human hepatoma cell lines were bought from the American Type Culture Collection (Manassas, Virginia, USA). Dt81Hepa1-6 cell line was derived from Hepa1-6 cells through passage in C57BL/6 mice . All cultures were maintained at 37C and 5% CO2. Cell lines were cultured in 0, 5.5 and 25?mM glucose DMEM supplemented with 10% FBS. All culture medium contained penicillin [100units/ml] and streptomycin [100g/ml]. If not stated otherwise, cells were seeded at 0.125M cells/cm2 for Hepa1-6, 0.25M cells/cm2 for Dt81Hepa1-6, 0.0625M cells/cm2 for Huh7 and 0.185M cells/cm2 for HepG2 to achieve 70% of cell confluence . Etomoxir [40 M] and sodium oxamate [100 mM] were used to inhibit fatty acid oxidation and glycolysis respectively [17,18]. Glucose uptake assay Following 30 minutes of glucose starvation, Hepa1-6, Dt81Hepa1-6, Huh7 and HepG2 cells were incubated for 45 minutes in presence of a fluorescent glucose analog, 2-NBDG, at increasing concentrations [0 to 100 M]. All subsequent steps were performed in the dark. The 2-NBDG reaction was stopped by washing cells with ice-cold phosphate-buffered saline (PBS). Glucose uptake was then quantified by measuring the fluorescent intensity of cells on a FACS BD LSRII flow cytometer (BD Biosciences, Mississauga, On, Canada). Data analysis was performed using FlowJo v10 (Tree Star, Ashland, Or, USA). Acquisition of fluorescent images was performed using a Leica Epifluorescence Microscope SP5 platform (Leica Microsystems, Richmond Hill, On, Canada). Quantitative analysis of 2-NBDG-labeled Hepa1-6 and Dt81Hepa1-6 cells was done using Fiji software (ImageJ, NIH, USA). qPCR gene expression analysis mRNA was isolated with TRIZOL (Invitrogen Burlington, On, Canada) according to the manufacturer instructions. 250ng of mRNA was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit. Quantitative Trolox PCR amplifications were performed using the QuantiTect SYBR Green PCR Kit in a Rotor-Gene 3000 Real-Time Thermal Cycler (Corbett Research, Sydney, Australia). For each gene tested, 35 amplification cycles at 59C (annealing) were used. The primer sequences are Trolox summarized in supplementary Table 1. Relative gene expression was evaluated using 3 reference genes: and . Relative gene expression was calculated using the delta delta CT method . HPLC analysis All metabolites described in this study were assessed using HPLC (Agilent 1200 HPLC system, Agilent Technologies Canada Inc., Mississauga, On, Canada) by the Metabolomic Core Facility of CRCHUM. Metabolic measurements were done on Hepa1-6 and Dt81Hepa1-6 cells after a 48 hours incubation in each indicated culture conditions. Culture cells (after removal of cell culture medium) were snap frozen in liquid nitrogen and kept at -80C until HPLC analysis. HPLC peak areas were used for quantification of identified metabolites. Total protein content (Bradford protein assay ), was used to normalize the metabolite quantification. Triglyceride assay Cellular TNF-alpha triglyceride (TG) content was measured on Hepa1-6 and Dt81Hepa1-6 cells after 48?hours of incubation in each indicated culture conditions. Cells were harvested on ice by scraping and washed with ice cold PBS. Samples were frozen over carbonated ice and kept at -80C. Lipids were extracted overnight from cell pellets (4C) with chloroform:methanol (2:1) (Folch extraction) . Organic phases (chloroform) were transferred into new glass tubes and dried under nitrogen.