Rapamycin (Sirolimus)

2 years -80 in solvent

Injection i.p.

U87-MG, T98G, and U373-MG

Dissolved in DMSO, final concentrations ca.25 uM

Dissolved in solvent solution (0.2% carboxymethylcellulose and 0.25% Tween-80 in sterile H2O)

Rapamycin inhibits endogenous mTOR activity in HEK293 cells with IC50 of ca.0.1 nM, more potently than iRap and AP21967 with IC50 of ca.5 nM and ca.10 nM, respectively. [1] In Saccharomyces cerevisiae, Rapamycin treatment induces a severe G1/S cell cycle arrest and inhibition of translation initiation to levels below 20% of control. [2] Rapamycin significantly inhibits the cell viability of T98G and U87-MG in a dose-dependent manner with IC50 of 2 nM and 1 uM, respectively, while displaying little activity against U373-MG cells with IC50 of >25 uM despite the similar extent of the inhibition of mTOR signaling. Rapamycin (100 nM) induces G1 arrest and autophagy but not apoptosis in Rapamycin-sensitive U87-MG and T98G cells by inhibiting the function of mTOR. [3]

72 hours

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 g/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 g/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.

Rapamycin (Sirolimus) is a specific mTOR inhibitor with IC50 of ~0.1 nM HEK293 cells.Rapamycin binds to FKBP12 and specifically acts as an allosteric inhibitor of mTORC1. Rapamycin is an autophagy activator and an immunosuppressant.