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Lactate was measured with a Lactate Assay Kit II (Bio Vision) according to the manufacturer's protocol. The glucose and lactate values were normalized to the protein concentration determined using a Bradford assay kit (Bio-Rad, Tokyo, Japan). Expression array analysis Total RNA was extracted from HCT116 and HT29 cells?cultured under 2DC or 3DC using Trizol. Gene expression arrays were conducted using a Whole Human Genome DNA microarray (4��44K) v2 (Agilent Technologies, Santa Clara, CA) and analyzed by Feature Extraction software (Agilent Technologies). Chromatin immunoprecipitation assay The cDNA encoding full-length MLX was cloned from HCT116 cells using PCR. Amplicons were then subcloned into a pcDNA3.1/V5/His TOPO vector (Invitrogen) under control of the CMV promoter. HCT116 cells were grown in 10-cm check details plates and transfected with pcDNA3.1/V5/His TOPO vector-MLX using FuGENE? HD Transfection Reagent (Promega). HCT116-MLX cells were seeded onto 15-cm plates and medium was replaced with RPMI1640 medium containing Megestrol Acetate 5.5 or 25?mmol/L glucose after cells became subconfluent. After 12?h incubation, cells were crosslinked by adding 1% formaldehyde in phosphate buffered saline (PBS) for 15?min at room temperature. Glycine was added at a final concentration of 0.125?mol/L for 5?min at room temperature to terminate the cross-linking reaction. Cells were harvested using a cell scraper after adding 2?mL cold PBS containing 1�� Protease Inhibitor Cocktail II. Nuclear extraction, sonication, immunoprecipitation, crosslink reversal, and DNA cleanup were performed using Millipore EZ-Magna ChIPTM A (Millipore, Billerica, MA) according to the manufacturer's protocol. Immunoprecipitation find more was performed with anti-His monoclonal antibody (Abcam, Cambridge, UK), mouse IgG, anti-tri-methyl-histone H3 (Lys 4) rabbit antibody or normal rabbit IgG (CST Japan, Tokyo, Japan). DNA fragments were quantified by real-time PCR using SYBR Premix Ex Taq (Takara) with the primers listed in Table S2. DNA eluted from the DNA�Cprotein complex before immunoprecipitation was used as ��input.�� The relative value of DNA fragments was calculated by extrapolation from a standard curve of input DNA dilutions. Statistical analysis The statistical significance of differences between values was assessed using the Mann�CWhitney U test. A value of P?
