Transient siRNA knockdown of MTHFD2 Depletion of endogenous MTHFD2 was achieved using three different siRNA oligonucleotides targeting MTHFD2 (Dharmacon). Nontargeting siRNA was used as a control (Dharmacon). Transfection was performed on log(phase) cells using 10 nM siRNA and INTERFERin transfection reagent (Polyplus-Transfection), following the manufacturer’s protocol. Briefly, cells were seeded 1 d before transfection and allowed to attach overnight. Control and targeting siRNAs were mixed with INTERFERin in serum-free medium, vortexed for 10 s and incubated at room temperature for 15 min to allow transfection complex formation. Cells were washed and changed to fresh complete medium before adding the transfection mix. The cells were incubated with the siRNAs at 37 °C with 5% CO2 in a humidified incubator until harvesting. Transfection was performed on log(phase) U2OS cells using 4 µg of plasmid DNA and jetPEI transfection reagent (Polyplus-Transfection), according to the manufacturer’s protocol. Successful and stable plasmid integration was maintained by culturing the cells in complete medium supplemented with 250 μg ml−1 of G418 disulfate (Sigma-Aldrich).