Cell culture and growth measurement

HEK293T, HeLa, HT-29, fibroblast, and myoblast cells were cultured at 37 °C in 5% CO₂ in DMEM (D5796, Sigma) supplemented with 10% or 20% FBS (Gibco). The viable cell numbers were counted by trypan blue staining assay.

Growth of HEK293T cells was measured using AlamarBlue (Invitrogen) in glucose medium (Wako DMEM 042–32255 supplemented with 10% FBS and 1 mg/mL glucose) or galactose medium (Wako DMEM 042–32255 supplemented with 10% FBS and 1 mg/mL galactose). Cells were inoculated at a density of 2.0 × 10³ cells/well on collagen I-coated 96-well plates (Iwaki). On each day of cell cultivation (days 0 to 6), 1/10 volume of AlamarBlue reagent was added to each well and incubated for 4 h; absorbance was then measured at 570 nm and 600 nm using a SpectraMax 190 microplate reader (Molecular Devices).

For growth measurement in non-bicarbonate medium, bicarbonate-free DMEM (D5648, Sigma) was supplemented with 10% FBS (Gibco), 30 mM HEPES (Rikaken), and 10 μg/mL each of nucleosides (adenosine, cytidine, guanosine, and uridine), and adjusted to pH 7.1 with NaOH. To add back bicarbonate, the non-bicarbonate medium was supplemented with 44 mM NaHCO₃, and adjusted to pH 7.2 with HCl. HEK293T cells precultured in DMEM were suspended in the non-bicarbonate medium. Cells were seeded at a density of 4 × 10³ cells/well on 96-well cell culture plates (TrueLine), and cultured at 37 °C under air (~0.04% CO₂) for the non-bicarbonate medium, and under 5% CO₂ for the bicarbonate-added back medium, respectively. On each day of cell culture, 1/10 volume of 1.4 mM resazurin sodium salt (Nacalai Tesque) dissolved in PBS was added to each well and incubated for 3 h. Fluorescence was then measured with excitation/emission at 550/600 nm using an Infinite 200 Pro M Plex (TECAN).

To estimate CO₂ effect on t⁶A frequency, HEK293T cells were cultured in the non-bicarbonate medium with or without 44 mM NaHCO₃ as described above, under 5% CO₂ or air (~0.04% CO₂), respectively, at 37 °C for 3 days. For mt-tRNA^Ser(AGY), prolonged cultivation was conducted as following. The cells were seeded in 15 cm dishes and pre-cultured to 70% confluence at 37 °C in 5% CO₂ in DMEM (D5796, Sigma) supplemented with 10% FBS (Gibco). Then, the cells were washed twice with non-bicarbonate medium consisted of bicarbonate-free DMEM (D5648, Sigma), 10% FBS (Gibco), 30 mM HEPES-NaOH (pH 7.4), and 10 μg/mL each of nucleotides (adenosine, cytidine, guanosine, and uridine) (final, pH 7.2), and then cultured with the same medium at 37 °C without CO₂ (air atmosphere) for 6 days. The medium was exchanged every 3 days. Total RNA was extracted, individual tRNAs were isolated, and tRNA modifications were analyzed by MS as described below.