The transfection was performed in six-well plates (Sarstedt 83.3920.300). A total of 2.5 × 105 cells were seeded per well in 3 mL of RPMI medium. Twenty-four hours after seeding, the cells were transfected using 1.5 µg of DNA per expression plasmid (combination of GFP-Tet3 and one of the Glud1 expression plasmids), 4 µL of jetPRIME, and 150 µL of jetPRIME buffer. In case of GFP-Tet3 only expressing cells, 1.5 µg of pESG-iba45 were co-transfected to keep the amount of transfected DNA constant. Six hours after transfection, the medium was changed and 10, 24, and 48 h after transfection, the cells were harvested and washed once with 1 mL of ice-cold PBS. Hundred microliters were used for GFP signal quantification of living cells using FACS (BD LSRFortessa; FSC 130 V, SSC 300 V, GFP 370 V log, 10,000 events per measurement), the rest was lysed and gDNA was isolated and analyzed by UHPLC-QQQ-MS as described previously using the Agilent 6400 Series Triple Quadrupole LC/MS System with the associated MassHunter Workstation Acquisition software and MassHunter Quantitative Analysis software45 (link). The hmdC/dN values were subsequently divided by the mean GFP signal to obtain hmdC/(dN*RFU).
When cells were fed with 4 mM DM-αKG, DM-αKG was added with the medium change 6 h after transfection.
When cells were fed with 4 mM DM-αKG, DM-αKG was added with the medium change 6 h after transfection.