Sponse elements in LDHB and LDHA promoters (A) Potential ERRa binding sites numbered relative to the transcription starting site (TSS) (B) Chromatin ImmunoPrecipitation (ChIP) assay for LDH promoters in XTC.UC1 cells using a polyclonal ERRa antibody. Chromatin was immunoprecipitated with the indicated antibody and submitted to quantitative PCR. Results 25033180 are expressed as fold change of enrichment compared to control IgG immunoprecipitated material. ERRa-IP was realized in duplicate and each sample was tested in triplicate for quantitative PCR. TFBS: transcription factor 
binding site. doi:10.1371/journal.pone.0058683.gERRa and Lactate Deshydrogenase B RegulationFigure 
3. ERRa Autophagy inhibits LDHB promoter activity. (A) Different construction of the human LDHB promoter reporter plasmid. (B) RO82W-1 cells were transfected with the indicated promoter constructs together with the expression plasmid of ERRa and/or PRC. Luciferase activity was determined 48 h after transfection and normalized against renilla luciferase activity. Results, presented in Relative Light Units (RLU), are the mean values6SD of three experiments performed in duplicate. *: p#0.05 versus cells transfected with plasmids controls and no ERRa or PRC. doi:10.1371/journal.pone.0058683.gReal-time quantification was performed in a 96-well plate using the IQ SYBR Green SuperMix and Chromo4 (Biorad). Data were normalized to b-globin as described elsewhere [6].Respiratory ParametersRespiratory parameters were investigated on intact cells from cultured cell lines and sample tissues by polarography, using a high-resolution Oroboros O2k oxygraph (Oroboros Instruments, Innsbruck, Austria) as described elsewhere [19,20]. The basal respiratory rate, defined as respiration in the cell-culture medium without additional substrates or effectors, was determined by measuring the linear rate of oxygen flux in intact cells (3.106 cells placed at 37uC in 2 ml Dulbecco’s inhibitor modified medium).were expressed as relative LDH to CS activities as an indicator of global cell metabolism. Lactate concentration in the culture media was determined by spectrophotometry using appropriate enzymatic kits (Boehringer Mannheim, Germany) on a Hitachi-Roche 917 (Roche Diagnostics GmbH Mannheim, Germany) and normalized to total cell numbers.Microarray AnalysiscDNA from RO82W-1 cells were hybridized in duplicate on human 4644,000 expression chips (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s recommendations. Data are available in the GEO database (GSE 37017). The Expression Analysis Systematic Explorer (EASE) and Gene Set analysis were used to determine the statistically over-represented and differentially expressed genes. Gene ontology enrichments in gene lists were searched for by means of the GOMiner. The most abundant gene ontology terms, representing at least 5 of the genes in the lists, with p values lower than 0.05, were considered for interpretation.Enzymatic ActivitiesThe activity of citrate synthase (CS), and Lactate Deshydrogenase (LDH) was measured spectrophotometrically (at 412 nm for CS and 340 nm for LDH) on cell lysates at 37uC in a cell buffer (250 mM saccharose, 20 mM tris[hydroxymethyl]aminomethane, 2 mM EGTA, 1 mg/ml bovine serum albumin, pH 7.2) using a Beckman DU 640 spectrophotometer (Beckman Coulter). Specific enzymatic activities were expressed in mIU (i.e. nanomoles of 5,5-dithiobis(2-nitrobenzoic acid)/min/mg portein for CS or nanomoles of NADH/min/mg protein for LDH).Sponse elements in LDHB and LDHA promoters (A) Potential ERRa binding sites numbered relative to the transcription starting site (TSS) (B) Chromatin ImmunoPrecipitation (ChIP) assay for LDH promoters in XTC.UC1 cells using a polyclonal ERRa antibody. Chromatin was immunoprecipitated with the indicated antibody and submitted to quantitative PCR. Results 25033180 are expressed as fold change of enrichment compared to control IgG immunoprecipitated material. ERRa-IP was realized in duplicate and each sample was tested in triplicate for quantitative PCR. TFBS: transcription factor binding site. doi:10.1371/journal.pone.0058683.gERRa and Lactate Deshydrogenase B RegulationFigure 3. ERRa inhibits LDHB promoter activity. (A) Different construction of the human LDHB promoter reporter plasmid. (B) RO82W-1 cells were transfected with the indicated promoter constructs together with the expression plasmid of ERRa and/or PRC. Luciferase activity was determined 48 h after transfection and normalized against renilla luciferase activity. Results, presented in Relative Light Units (RLU), are the mean values6SD of three experiments performed in duplicate. *: p#0.05 versus cells transfected with plasmids controls and no ERRa or PRC. doi:10.1371/journal.pone.0058683.gReal-time quantification was performed in a 96-well plate using the IQ SYBR Green SuperMix and Chromo4 (Biorad). Data were normalized to b-globin as described elsewhere [6].Respiratory ParametersRespiratory parameters were investigated on intact cells from cultured cell lines and sample tissues by polarography, using a high-resolution Oroboros O2k oxygraph (Oroboros Instruments, Innsbruck, Austria) as described elsewhere [19,20]. The basal respiratory rate, defined as respiration in the cell-culture medium without additional substrates or effectors, was determined by measuring the linear rate of oxygen flux in intact cells (3.106 cells placed at 37uC in 2 ml Dulbecco’s modified medium).were expressed as relative LDH to CS activities as an indicator of global cell metabolism. Lactate concentration in the culture media was determined by spectrophotometry using appropriate enzymatic kits (Boehringer Mannheim, Germany) on a Hitachi-Roche 917 (Roche Diagnostics GmbH Mannheim, Germany) and normalized to total cell numbers.Microarray AnalysiscDNA from RO82W-1 cells were hybridized in duplicate on human 4644,000 expression chips (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s recommendations. Data are available in the GEO database (GSE 37017). The Expression Analysis Systematic Explorer (EASE) and Gene Set analysis were used to determine the statistically over-represented and differentially expressed genes. Gene ontology enrichments in gene lists were searched for by means of the GOMiner. The most abundant gene ontology terms, representing at least 5 of the genes in the lists, with p values lower than 0.05, were considered for interpretation.Enzymatic ActivitiesThe activity of citrate synthase (CS), and Lactate Deshydrogenase (LDH) was measured spectrophotometrically (at 412 nm for CS and 340 nm for LDH) on cell lysates at 37uC in a cell buffer (250 mM saccharose, 20 mM tris[hydroxymethyl]aminomethane, 2 mM EGTA, 1 mg/ml bovine serum albumin, pH 7.2) using a Beckman DU 640 spectrophotometer (Beckman Coulter). Specific enzymatic activities were expressed in mIU (i.e. nanomoles of 5,5-dithiobis(2-nitrobenzoic acid)/min/mg portein for CS or nanomoles of NADH/min/mg protein for LDH).
