Animals
All procedures were conducted in accordance with approved animal protocols by the McGill University Animal Care Committee. Wild-type mice (C57BL/6J) and muscle creatine kinase (mck)-PGC-1α transgenic mice (C57BL/6-Tg(Ckm-Ppargc1a)31Brsp/J) as described in [14] were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animals used were female and were sacrificed at approximately 12 weeks of age.
Tissue culture
Stable cell lines Control-1, PGC-1α-1.1, and PGC-1α-1.2 have been described in [13]. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 10% FBS, 10 μg/mL insulin, 20 mM HEPES, penicillin/streptomycin, 1 μg/mL puromycin, 400 μg/mL G418, at 37°C and 5% CO2. SK-BR-3 and BT-474 were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Hypoxia experiments were performed in a Thermo Scientific (Rockford, IL, USA) Heraeus HERAcell 150 set at 1% O2 and 5% CO2. Pharmacological inhibition of ERRα was performed using Compound 29 (C29) at 5 μM (0.1% DMSO). C29 was synthesized according to [15].
Gene expression
Total RNA from cultured cells, grown to 50% to 60% confluence in 35 mm plates, was extracted using the Aurum Total RNA Mini Kit (Bio-Rad, Mississauga, Canada) and was reverse transcribed with iScript cDNA Synthesis kit (Bio-Rad). mRNA expression analyses by real-time PCR were performed using iQ SYBR Green Supermix (Bio-Rad) and gene-specific primers with the MyiQ2 Real-Time Detection System (Bio-Rad). Values were normalized to TATA binding protein (Tbp) for murine cell lines or beta-2 microglobulin (B2M) for human cell lines.
Chromatin immunoprecipitation
Chromatin was prepared from PGC-1α-1.1 cells, using approximately 107 cells per chromatin immunoprecipitation (ChIP). ChIP was performed as described previously [16] using a specific anti-PGC-1α antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, catalog #sc-13067) or specific anti-ERRα antibody (Epitomics, Burlingame, CA, USA, catalog #2131-1). Quantification of PGC-1α or ERRα ChIP enrichment was performed using real-time PCR using the LightCycler 480 instrument (Roche, Mississauga, Canada) and specific primers for genomic regions. ChIP enrichment was normalized against the control region and further normalized against the IgG control.
Immunoblotting
Total proteins from cultured cells or muscle tissue were extracted with lysis buffer (50 mM Tris–HCl pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) with inhibitors (2 μg/mL pepstatin, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 0.2 mM phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate) and quantified with the Bio-Rad Protein Assay kit (Catalog #500-0006). The blots were incubated according to the manufacturer's instructions with the following primary antibodies: Glud1 (GeneTex, Irvine, CA, USA, GTX88164), Gls (Abcam, Toronto, Canada, AB93434), Got1 (GeneTex, GTX88903), Got2 (GeneTex, GTX88925), Hsp90 (Santa Cruz Biotechnology, sc-101494), and Actin (Santa Cruz Biotechnology, sc-1616) and with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Mississauga, Canada, or Santa Cruz Biotechnology). The results were visualized using Western Lightning Plus-ECL (Perkin Elmer, Waltham, MA, USA) or SuperSignal West Femto ECL (Pierce, Rockford, IL, USA). Densitometry analyses were conducted using ImageJ software (NIH).
siRNA
BT-474 cells were subjected to either 10 nM control siRNA (Dharmacon, Rockford, IL, USA, D-001810-10-05) or a combined 10 nM pool of four siRNA specifically targeting PPARGC1A (Qiagen, Germantown, MD, USA, FlexiTube GeneSolution GS10891). Cells were transfected using calcium phosphate (CaPO4) and incubated for 120 h before extraction.
14C-lipid incorporation assay
Cells were seeded at a density of 400,000 cells per 35 mm plate in standard growth media and conditions. After 24 h, the medium was supplemented with trace amounts of [U-14C]-glutamine (0.094 μCi/mL) and the cells were further incubated either in normoxia (21% O2) or hypoxia (1% O2) for 24 h (Control-1 and PGC-1α-1.1) or 48 h (BT-474). Individual experiments were conducted in triplicate with additional plates for cell counting. Cells were washed with PBS, collected using 0.5% Triton X-100, and stored at −80°C. After thawing, lipids were extracted using chloroform/methanol; the lipid-rich chloroform phase was dried under air and resuspended in 150 μL chloroform. Scintillation counts were measured over 5 minutes per sample using a MicroBeta2 scintillation counter (Perkin-Elmer) and Eco-Lume reagent (MPBio, Solon, OH, USA). Counts were normalized for cell number.
Glutamine uptake
Cellular glutamine uptake was measured using the BioProfile 400 analyzer (Nova Biomedical Corp., Waltham, MA, USA). Following growth under normoxic (21% O2) or hypoxic (1% O2) conditions, media were removed and maintained on ice until analysis. Glutamine uptake was calculated as the difference in glutamine content between culture media and unseeded media incubated in parallel plates and normalized for cell count.
Mass isotopomer distribution analysis
The metabolomics analyses by GC/MS were adapted from [17]. Additional details are provided in Additional file 1.
The Cancer Genome Atlas analyses
Breast cancer data, clinical data, and survival data for 732 patients were obtained from The Cancer Genome Atlas (TCGA) [18]. RNAseq data were downloaded from TCGA web portal using the reads per kilobase of exon model per million mapped reads (RPKM) quantification [19]. RPKM data was log2 transformed and expression values lower than 0.25 RPKM were removed. To generate an overall pathway score, all nine genes in the glutamine metabolism pathway were Z-transformed (mean 0 and standard deviation of 1). Mean of zero corrects for the fact that the genes may be expressed at different levels. Transforming the standard deviation to 1 causes the genes to change in the same magnitude, so we can see if the overall pathway is over/underexpressed. If the genes are expressed at different levels, the changes in the pathway will be dominated by the genes that were originally expressed at higher levels. We used the median of Z-scores to generate a pathway score, which was used in correlation and survival analysis.
Statistical analyses
All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., CA, USA), Microsoft Excel (Microsoft Corporation, CA, USA) or Matlab (The Mathworks, Inc., MA, USA). Survival analysis was calculated using an implementation of Kaplan-Meir log rank testing from Matlab Exchange [20].
Additional methods
Additional methods are provided in Additional file 1 and in Additional file 2: Table S1.