Glutamine

Pre-adipocyte 3T3-L1 cells were grown in DMEM medium supplemented with 10% FBS (Gibco), 2 mM glutamine (Wisent) and 1% Penicillin/Streptomycin (Biobasic). For adipogenic differentiation of 3T3L1, the cells were plated at confluency and media was changed to induction media containing 10% FBS, 1% Penicillin/Streptomycin, 1 μM Dexamethasone, 1 μg/ml Insulin and 500 μM IBMX (Sigma). Two days post-induction, the medium was changed to maintenance media containing 10% FBS (Gibco), 1% Penicillin/Streptomycin (Biobasic), 1 μg/ml Insulin. After 3 days post-induction, 10,000 cells were plated on homemade chambers for sorting.
Mouse Embryonic Stem cell (mES) culture mES cells were grown in DMEM medium supplemented with 15% FBS (embryonic stem cell qualified, Wisent), 1 X non-essential amino acids (Sigma), 100 μM 2-Mercaptoethanol (Gibco), 1000 Units/mL Leukemia inhibitory factor (LIF, Stemcell), 2 mM glutamine (Wisent) and 1% Penicillin/Streptomycin (Biobasic) on 0.1% porcine gelatin-coated plastic dishes (Sigma). About 10,000 cells were plated for sorting as above.

Opti Eagle’s minimum essential media (Opti-MEM), penicillin, streptomycin, fungizone, glutamine, and fetal calf serum (FCS) were purchased from Wisent (Montreal, QC). Ficoll–Paque was purchased from Amersham Biosciences (Montreal, QC). Human recombinant (hr) M-CSF, and hrGM-CSF were purchased from R&D (R&D Systems, Minneapolis, MN); soluble hrRANKL was produced in our laboratory. Rabbit polyclonal antibodies against human PIDD (#ab78389), Galectin 8 (#ab41649), and RHOT1 (#ab83779) were purchased from Abcam (Cambridge, MA); rabbit polyclonal antibodies against human TBC1D25 (OATL1) (#HPA029197), and USP4 (#U0635) were purchased from Sigma-Aldrich (St. Louis, MO), and fluorescent Alexa antibodies, Di Aminido Phenyl lndol (DAPI), and siRNA from Invitrogen (Burlington, ON).

All cells were in the D-Mel (d.mel-2) background and were cultured in Express Five medium (Invitrogen) supplemented with glutamine, penicillin and streptomycin (Wisent). Transfections were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) following the manufacturer's instructions. All stable cell lines were selected in medium containing 20 µg ml⁻¹ blasticidin. While inducible pMT-based vectors contain the blasticidin resistance gene, pAc5-based vectors were co-transfected with pCoBlast to confer blasticidin resistance to the cells. Expression of the copper-inducible transgenes was induced with CuSO₄ (300 µM unless otherwise indicated) for at least 8 h before experiments.
For RNA interference, dsRNAs were generated from PCR amplicons using a T7 RiboMAX kit (Promega). dsRNA derived from the bacterial kanamycin resistance gene was used as a non-target control. Twenty milligrams of dsRNA was transfected in 1X10⁶ cells in a well of a 6-well plate using Transfast transfection reagent according to the manufacturer's protocol.