Rapamycin (AY-22989)

HEK293 cells are plated at 2-2.5×10⁵ cells/well of a 12-well plate and serum-starved for 24 hours in DMEM. Cells are treated with increasing concentrations of Rapamycin (0.05-50 nM) for 15 minutes at 37 °C. Serum is added to a final concentration of 20% for 30 minutes at 37 °C. Cells are lysed, and cell lysates are separated by SDS-PAGE. Resolved proteins are transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of p70 S6 kinase. Data are analyzed using ImageQuant and KaleidaGr

Cells are exposed to various concentrations of Rapamycin for 72 hours. For the assessment of cell viability, cells are collected by trypsinization, stained with trypan blue, and the viable cells in each well are counted. For the determination of cell cycle, cells are trypsinized, fixed with 70% ethanol, and stained with propidium iodide using a flow cytometry reagent set. Samples are analyzed for DNA content using a FACScan flow cytometer and CellQuest software. For apoptosis detection, cells are stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique using an ApopTag apoptosis detection kit. To detect the development of acidic vesicular organelles (AVO), cells are stained with acridine orange (1 μg/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 μg/mL) for 15 minutes, removed from the plate with trypsin-EDTA, and analyzed using the FACScan flow cytometer and CellQuest software. To analyze the autophagic process, cells are incubated for 10 minutes with 0.05 mM monodansylcadaverine at 37 °C and are then observed under a fluorescence microscope.