Sodium pyruvate 100x

To label newly synthesized mitochondrial DNA-encoded proteins, cells were seeded into a six-well dish at 80–90% confluency. First, two washing steps of 5 min each in methionine/cysteine-free DMEM were performed. Subsequently, cells were incubated with fresh methionine/cysteine-free DMEM supplemented with Glutamax 100X (Gibco), sodium pyruvate 100X (Gibco), 10% dialysed FBS and 100 µg ml⁻¹ emetine (Sigma-Aldrich) for 20 min at 37 °C. Labelling was performed with the addition of 166,7 µCi ml⁻¹ of EasyTag EXPRESS [³⁵S] protein labelling mix (methionine and cysteine) (Perkin Elmer) for 30 min at 37 °C. Following labelling, cells were washed with 1 ml of PBS three times and the final pellets were collected by centrifugation. Cells were lysed in 1× PBS-PIC with the addition of 50 units of benzonase (Life Technologies) with incubation on ice for 20 min, followed by the addition of SDS to 1% final concentration. and further incubation on ice for 30 min. After cell lysis, 30 µg total protein was separated on Bolt 12% Bis-Tris Plus (Invitrogen) SDS–PAGE gels. Gels were then incubated in Imperial Protein Stain (Thermo Fisher) for 1 h and with fixing solution (20% methanol, 7% acetic acid and 3% glycerol) for 1 h. Next, gels were vacuum-dried at 65 °C for 2 h. The resultant gel was exposed to storage phosphor screens and visualized with Typhoon FLA 7000 Phosphorimager.

Glioma stem cell line, GSC23 was kindly provided by the MD Anderson Cancer Center, University of Texas^{35 (link)}. HMSCs were bought from ScienCell Research Laboratories (Sciencell, Cat. #7500, CA, USA). Similar to previously reported methods^{36 (link)}, GSC23 cells were transfected with red fluorescence protein (RFP) gene, and HMSCs were transfected with green fluorescence protein (GFP) gene. In another method, GSC23 cells were also transfected with a lentiviral vector GV348 (sequence element: Ubi-MCS-SV40-puromycin) containing LOXP-STOP-LOXP-RFP gene, and MSCs were transfected with a lentiviral vector GV348 containing a CRE enzyme gene. The RFP, GFP and CRE-LOXP lentiviral vectors were packaged by Shanghai Genechem Co., Ltd (Shanghai, China). MSCs were cultured in Mesenchymal Stem Cell Medium (MSCM, Sciencell, Cat. #7501). GSC23 cells were maintained in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12), containing 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), B27 supplement (50X), 2 mmol/l L-glutamine (100X), MEM vitamin solution (100X) and 100 mM sodium pyruvate (100X) (all from Gibco, Carlsbad, CA, USA).