Fla 7000 fluorimager

Primer extension was conducted basically as described (27 (link),28 (link)). The 5΄ ³²P-labeled primer (0.1 pmol) was incubated with 3.125 μg total RNA in a 5 μl mixture containing 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 2 min at 80°C, followed by cooling to room temperature. Subsequently, 0.5 μl (100 units) SuperScript III (Invitrogen) was added to 4.5 μl of the premixed solution [2 μl 5 × FS buffer (Invitrogen), 0.25 μl of 1.5 mM d/ddNTP mix, 0.75 μl ddH₂O and 1.5 μl of 25 mM MgCl₂]. The d/ddNTP mix consisted of dTTP, dGTP and ddCTP for mt-tRNA^Lys; dTTP, dGTP and ddATP for mt-tRNA^Arg; and dATP, dTTP, dCTP and ddGTP for mitochondrial 16S rRNA. The reaction was carried out for 1 h at 47°C for mt-tRNAs and 55°C for mitochondrial 16S rRNA. The reaction was stopped by addition of 0.5 μl of 4 M NaOH and boiling for 5 min at 95°C, and then neutralized by addition of 4.5 μl of 1 M Tris–HCl (pH 5.0). The cDNAs were resolved by 20% denaturing PAGE. The gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) and visualized on a FLA-7000 fluorimager (Fujifilm).

Pulse-labeling of mitochondrial protein synthesis was performed as described previously (34 (link)). Myoblasts (4.0 × 10⁵) were inoculated on 6-well plates and cultured at 37°C for 10 min with 1 ml of l-glutamine/l-cysteine-free DMEM (21013-024, Gibco) containing 10% dialyzed FBS (Gibco), 2 mM l-glutamine (Sigma) and 10 mM taurine, followed by addition of 50 μg/ml emetine to inhibit cytoplasmic protein synthesis and incubation for 10 min. Then, the cells were pulsed with 7.4 MBq (0.2 mCi) of [³⁵S] Met and [³⁵S] Cys (EXPRE³⁵S³⁵S Protein Labeling Mix, [³⁵S]-, PerkinElmer) and incubated at 37°C for 1 h to specifically label mitochondrial translation products. The medium was changed to DMEM-high glucose (D5796, Sigma-Aldrich) containing 10% dialyzed FBS (Gibco) and 10 mM taurine, and incubated for 10 min. Cell lysates (25 μg total proteins) were resolved by Tricine-SDS-PAGE (16.5%), and the gel was CBB-stained (CBB stain one, #04543-51, Nacalai Tesque) and dried on a gel drier (AE-3750 RapiDry, ATTO). The dried gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) to visualize the radiolabeled bands using the FLA-7000 fluorimager (Fujifilm).

HEK293T WT, ALKBH1 KO and NSUN3 KO cells (2.0 × 10⁴) were seeded on 10 cm dishes and cultured overnight. Cells were washed with L-Met-, L-Gln- and L-Cys-free medium (Sigma-Aldrich) supplemented with 2 mM L-Gln, 10% FBS and 1 mM sodium pyruvate. To inhibit cytoplasmic protein synthesis, 50 μg/ml emetine was added to the medium and incubated for 10 min. The cells were further supplemented with 8.14 MBq (0.22 mCi) of [³⁵S] Met/[³⁵S] Cys (EXPRE³⁵S³⁵S Protein Labeling Mix, [³⁵S]-, PerkinElmer) and incubated for 1 h to label proteins translated in mitochondria. Then, the medium was replaced with cold medium (DMEM with 10% FBS) and chased for 10 min, and the cells were harvested by centrifugation at 1500 × g for 10 min at 4°C. The cell lysates were resolved by Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (16.5%), and the gel was coomassie brilliant blue (CBB)-stained and dried by a gel drier (AE-3750 RapiDry, ATTO). The dried gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) and visualized on a FLA-7000 fluorimager (Fujifilm).