Otope ratio 13C enrichment of (a) lactate, (b) succinate and (c

Otope ratio 13C PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28393612 enrichment of (a) lactate, (b) succinate and (c) glutamate analyzed by GC-MS of the aqueous fraction from control and PPARg agonist-treated 3T3-L1 cells incubated with 1-13C-glucose. (d) M+1/M isotope ratio 13C enrichment of arachidate analyzed by GC-MS of the organic fraction from control and PPARg agonist-treated 3T3-L1 cells incubated with 1-13C-glucose. *P < 0.05. The metabolites have been mapped to the glycolysis, TCA cycle and fatty acid synthesis metabolic pathways. An upward pointing arrows indicates a metabolite increased in 13C enrichment by PPARg activation, and a downward pointing arrows indicates a metabolite decreased in 13C enrichment by PPARg activation. Parent ions were used to calculate ion ratio. Additional file 2: Figure S2 - the average integrated area of the two-dimensional HSQC-NMR organic fraction glycerol peak. (a) A graph of the average integrated area of the two-dimensional HSQC-NMR organic fraction glycerol peak (13C chemical shift 62.04) from control and 1 M PPAR agonist-treated 3T3-L1 adipocytes incubated in 1-13C glucose. (b) A graph of the average integrated area of the twodimensional HSQC-NMR organic fraction glycerol peak (13C chemical shift 62.17) from control and 1 M PPAR agonist-treated 3T3-L1 adipocytes incubated in 1-13C glucose. (c) A graph of the average integrated area of the two-dimensional HSQC-NMR organic fraction esterified glycerol peak from control and 1 M PPAR agonist-treated 3T3-L1 adipocytes3T3-L1 adipocytes were cultured and then treated with either the PPAR agonist, the PPARg agonist or vehicle control as described above (n = 6 independent individual hybridizations for each treatment group). RNA was extracted using RNeasy (Qiagen GmbH, Hilden, Germany). Approximately 5 mg of cells was used per sample for RNA isolation. Procedures were carried out according to the manufacturer's instructions. Extracted RNA was quantified and its purity assessed using a Nanodrop ND-1000 Spectrometer (Nanodrop Technologies PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28560407 Inc., Wilmington, NC, USA) to measure the absorbance at 260 nm and the A260/A280 ratio, SB-334867 respectively. Illumina Infinium Gene Expression BeadArrays (Illumina Inc., San Diego, CA, USA) were used to perform transcriptomics. A mouse WG6 array platform was used with 45,281 probes. Analysis was performed with R/BioConductor version 2.5. The R package lumi [37] was used with the detection P-value threshold set to 0.01. Probes were required to be successfully detected (P-Roberts et al. Genome Biology 2011, 12:R75 http://genomebiology.com/2011/12/8/RPage 18 ofincubated in 1-13C glucose. *P < 0.05, ***P < 0.005. Parent ions were used to calculate ion ratio. Additional file 3: Figure S3 - M+1/M isotope ratios. (a, b) The M+1/M isotope ratio 13C enrichment of (a) glutamate and (b) isocitrate analyzed by GC-MS of the aqueous fraction from control and PPARg agonisttreated 3T3-L1 cells incubated with 13C-U-palmitate. (c-e) Graphs showing the isotope ratio 13C enrichment of myristate (c), arachidate (d) and palmitate (e) analyzed by GC-MS of the organic fraction from control and PPARg agonist-treated 3T3-L1 cells incubated with 13C-U-palmitate. The metabolites have been mapped to the TCA cycle and fatty acid boxidation/synthesis metabolic pathways. Red indicates a metabolite increased in 13C enrichment by PPARg activation. Blue indicates a metabolite decreased in 13C enrichment by PPARg activation. *P < 0.05, **P < 0.01,***P < 0.005. Parent ions were used to calculate ion ratio. Additi.