TEM Analysis

At the end of each isotope-labeling incubation, cells were collected and centrifuged at 2,700 rpm for 10 min at room temperature. One microliter of the pellet was mixed with 1 μL of 20% bovine serum albumin and transferred to a formvar-coated 100 mesh TEM grid. After removing the excess liquid with a filter paper, the grids were frozen in liquid ethane cooled with liquid nitrogen. Freeze-substitution was carried out in a 2% mixture of OsO₄ in 100% acetone (v/v) sequentially at three temperatures: −90°C (for 96 h), −20°C (for 24 h), and 4°C (for 10 h). Temperature was increased at a rate of 5°C h^–1 (from −90 to −20°C) and 3°C h^–1 (from −20 to 4°C). After freeze-substitution, the samples were washed three times in acetone and infiltrated sequentially in a 2:1, 1:1, and 1:2 (v/v) mixture of acetone and low-viscosity Spurr resin (EMS) for 1 h in each step. Finally, the samples were incubated overnight in a 100% resin, transferred to embedding molds, and allowed to polymerize. Thin sections (200 nm) were cut with a diamond knife, placed on Cu-indexed TEM grids (rinsed in 30% ethanol), and contrasted for 20 min in saturated ethanolic uranyl acetate (EMS, Hatfield, United States; concentration 13 g/100 mL 50% ethanol; solution filtered before use through a 0.45 μm pore size filter). Images were taken using a JEOL 1010 TEM at 80 kV.