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In Vitro : Rapamycin inhibits endogenous mTOR activity in HEK293 cells with an IC50 of 0.1 nM, which is more effective than iRap and AP21967 with IC50 of 5 nM and 10 nM, respectively [1]. Rapamycin exerts its anti-tumor effect on malignant glioma cells by inducing autophagy and demonstrates that disruption of the PI3K/Akt signaling pathway in malignant glioma cells can greatly enhance the effectiveness of mTOR inhibitors. . Rapamycin inhibits cell viability in all three cell lines in a dose-dependent manner, but their sensitivity changes. The IC50 levels of T98G, U87-MG and U373-MG cells were 2 nM, 1 μM and >25 μM [3], respectively.
In Vivo : Rapamycin (ip, 1.5 mg / kg) treatment almost completely prevented hypertrophy of the diaphragm weight and fiber size at 7 and 14 days [4]. WT or LS / + mice were treated daily with rapamycin (2 mg / kg body weight, intraperitoneal injection) for 4 weeks, then the same dose was injected weekly for another 4 weeks. The abnormal fetal gene expression profile of rapamycin-treated LS/+ mouse hearts was significantly reversed [5].
Kinase Assay : HEK293 cells were seeded in 2-well plates at 2-2.5 x 105 cells per well and serum starved for only 24 hours in DMEM. Cells were mock treated or treated with rapamycin (0.05-50 nM), iRap (0.5-500 nM) or AP21967 (0.5-500 nM) for 15 minutes at 37 °C. Serum was added to a final concentration of 20% at 37 ° C for 30 minutes. Cells were lysed and cell lysates were separated by SDS-PAGE. The isolated protein was transferred to a PVDF membrane and immunoblotted with a phospho-specific primary antibody against Thr389 of p70 S6 kinase. Data were analyzed using ImageQuant and KaleidaGraph [1].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Cell Assay : HL-60, NB4, U937, KG-1 and K562 cells were routinely passaged in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 U / mL penicillin and 50 μg /mL streptomycin. At 37 ° C, 5% CO 2 humidified atmosphere. For the experiment, the exponentially growing cells were harvested by centrifugation and resuspended in fresh medium containing 10% FBS. Cells were seeded at BD Falcon six-well plates at an initial cell density of 2 x 10 5 /mL in the presence of various concentrations of DMSO or 1 μMATRA. Rapamycin (20 nM) was added 20 minutes before the differentiation agent. On day 2, 0.3 mL of fresh medium was added to each well. Viable cells were assayed by trypan blue exclusion and quantified using a hemocytometer [2].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Animal Administration : Rat [4]
Female Sprague-Dawley rats (250-275 g), adult female SD rats (225-250 g) were randomly assigned to treatment or vehicle groups such that the average starting body weight of each group was equal. The drug treatment begins on the day of surgery or the first day of reloading after 14 days of withdrawal. Rapamycin was delivered once daily by intraperitoneal injection at a dose of 1.5 mg / kg and dissolved in 2% carboxymethylcellulose. CsA was delivered by subcutaneous injection once a day at a dose of 15 mg / kg, dissolved in 10% methanol and olive oil. FK506 was delivered by subcutaneous injection once daily at a dose of 3 mg / kg, dissolved in 10% ethanol, 10% cremophor and saline.
Mouse [5]
The rapamycin was dissolved in ethanol at a concentration of 20 mg / mL, sterilized by filtration, resuspended in a carrier (0.25% PEG, 0.25% Tween-80) at a concentration of 1 mg / mL, and intraperitoneally injected. (2 mg / kg body weight) for 4 weeks per day or 4 weeks per day, followed by another 4 weeks per week. The injections were started at 8 weeks (before hypertrophy) or 12 weeks (indicated to determine hypertrophy), and the mice were evaluated after 4 weeks of treatment or after 8 weeks of treatment, as detailed in the results and legends. . As a control, WT and LS / + mice were treated with vehicle only.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Rapamycin biological activity and experimental methods
Rapamycin is a specific mTOR inhibitor with an IC50 of 0.1 nM