Hygromycin selection

TF-1 cells (#CRL-2003, ATCC; purchased 2018; authentication and Mycoplasma not tested) were cultured in RPMI 1640 medium with 10% FBS and 2 ng/ml recombinant human GM-CSF (R&D Systems). MOLM14 (from D. Gary Gilliand’s lab; authentication and Mycoplasma not tested) cells were cultured in RPMI 1640 medium with 10% FBS. HEK293T (from D. Gary Gilliand’s lab; authentication and Mycoplasma not tested) was cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% FBS. Cells were cultured at 37 with of 5% CO2. Stable overexpression of IDH2R140Q variants in TF-1 cells was conducted by using pLVX-IRES-Hyg vector harboring FLAG-tagged IDH2 R140Q, R140Q/K413R, or R140Q/K413Q. Briefly, to produce lentivirus, each construct was co-transfected into HEK293T cells using Lenti-X™ Packaging Single shots (Clontech) according to the manufacturer’s instructions. Lentivirus-containing supernatant medium was collected 48 hours after transfection, filtered before adding to the indicated host cell lines. 24 hours after infection, target cells were subjected to hygromycin selection (Invitrogen). The overexpression of proteins was confirmed by Western blotting using antibodies against IDH2.

Stable overexpression of IDH1 WT and mutants in A549 was conducted using retroviral vectors harboring RNAi-resistant FLAG-tagged IDH1 WT, and RNAi-resistant FLAG-tagged IDH1 Y42F/Y391F mutant. Briefly, to produce retrovirus, each construct was co-transfected with 0.1 μg VSVG, 0.9 μg EcoPak packaging plasmid, and 1 μg envelope plasmid (Addgene) into HEK293T cells seeded in 6 well plate using TransIT®-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions. Retrovirus-containing supernatant medium was collected 48 hours after transfection and filtered by 0.45 μm filter before addition to the indicated host cell lines with 3 μL 10 mg/ml polybrene(1 mol/L HEPES was used to adjust the pH to 7.4 in culture medium). Twenty-four hours after infection, target cells were subjected to hygromycin selection (Invitrogen). The overexpression of proteins was confirmed by Western blotting using antibodies against IDH1.