6 well plate

Normal human BSM cells (hBSMCs; Cambrex Bio Sience Walkersville,
Inc., Walkersville, MD, USA) were maintained in SmBM medium supplemented with 5%
fetal bovine serum, 0.5 ng/ml human epidermal growth factor (hEGF), 5 µg/ml
insulin, 2 ng/ml human fibroblast growth factor-basic (hFGF-b), 50 µg/ml
gentamicin and 50 ng/ml amphotericin B. Cells were maintained at 37 °C in a
humidified atmosphere (5% CO₂), fed every 48–72 h, and passaged when
cells reached 90–95% confluence. Then the hBSMCs (passages 4–7) were seeded in
6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at density of
3,500 cells/cm² and, when 80–85% confluence was observed, cells were
cultured without serum for 24 h before the addition of PGD₂. At the
indicated time after the treatment with PGD₂, hBSMCs were immediately
collected and disrupted with 1× SDS sample buffer (150 µl/well), and used for
Western blot analyses.

Cells were seeded on 6-well plates (Becton Dickinson [Corning], Franklin Lakes, NJ, USA), 24 h prior to the experiment (2 mL, 1 × 10⁵ cells/mL, incubation at 37°C, 5% CO₂). After washing the cells with 1 mL of phosphate-buffered saline (PBS (−)), the cells were incubated with medium that did not contain FBS and in the presence of the MITO-Porter (pre-WT-tRNA^Phe) (6.75 nM RNA [300 ng RNA]) for 3 h for direct mitochondrial transfection. MITO-Porter (RP-WT-tRNA^Phe-MRP), MITO-Porter (tRK1-WT-tRNA^Phe), or MITO-Porter (tRNA^Phe (yeast)) were also added to the cells at similar way (6.75 nM RNA). For allotropic expression, pCMV-WT-tRNA^Phe, pCMV-RP-WT-tRNA^Phe-MRP, pCMV-tRK1-WT-tRNA^Phe, or pCMV-WT-tRNA^Phe (yeast) were transfected to cells using LFN 2000, according to the manufacturer’s recommended protocol (1 μg). After the incubation, the medium was replaced with complete medium containing FBS and further incubated at 37°C, 5% CO₂, for 45 h or 93 h. After washing the cells with 1 mL PBS (−), they were treated with 500 μL of PBS (−) containing trypsin to remove cells. After incubation of the cells at 37°C, 5% CO₂, for 3 min, 1 mL of complete medium was added to the cells, followed by centrifugation at 700 × g at 4°C for 3 min. The supernatant was removed to obtain the collected cells (step 2).

To investigate the radiosensitivity of the cells, we performed colony-formation assays. Briefly, 5 × 10⁵ cells were seeded onto a T-25 flask (25 cm², FALCON Becton Dickinson, Franklin Lakes, NJ) and were maintained at 37°C in a humidified atmosphere with 5% CO₂ overnight. The next day, the medium was changed for fresh medium without FBS, and recombinant IL-6 was added 6 h later. After incubation for 24 h, the cells were irradiated with 6 Gy of γ-rays. The cells were trypsinized, and detached cells were collected and cultured in 6-well plates (Becton Dickinson), with an appropriate cell count depending on the survival rate (non-irradiated: 100, 6 Gy: 1000) for 10–12 days. After culturing, the plates were fixed with methanol and stained with 5% Giemsa solution. Colonies consisting of at least 50 cells were counted. The survival fraction was calculated from the plating efficiency.