Metabolic Reprogramming of SHSY5Y and 293LTV Cells

SHSY5Y cells were purchased from ECACC (94030304) and cultured in DMEM containing 4 mM glutamine and 5.5 mM glucose, or 10 mM galactose, respectively. 293LTV cell line was purchased from Cell Biolabs, Inc. (LTV-100) and cultured in DMEM containing 4 mM glutamine and 5 mM glucose. The GCN5L plasmid was a kind gift from Dr. Sack’s laboratory^{19 (link)}. The IDH2 WT and IDH2 K413Q plasmids were a kind gift from the laboratory of Dr. Denu^{13 (link)}. Stable cell lines were generated by treatment with G418 (200 µg/ml) after transfection. SIRT3 and SIRT3 H248Y plasmids were purchased from Addgene (#24924, #24917 respectively) and respective ORFs subcloned into the p3XFLAG-CMV14 vector (Sigma-Aldrich). Vector DNA was transfected using Lipofectamine 2000 (ThermoFisher Scientific). For RNA silencing, the predesigned Mission siRNAs (Sigma-Aldrich) were transfected using Lipofectamine RNAiMAX (ThermoFisher Scientific). The decline of RNA was verified using real-time PCR and data calculated using Double Delta Ct analysis.