All experimental animal protocols were approved by the Yale University Institutional Animal Care and Use Committee. Rapamycin (sirolimus; EMD Millipore) and MEK inhibitor PD0325901 (Selleckchem) were diluted in ethanol and mixed with an equal volume (30 µl) of Cremophor EL (Sigma-Aldrich). These drug mixtures were dissolved in Dulbecco’s PBS at a final volume of 0.2 ml/mouse and administered via intraperitoneal injection. Female C.B-17 SCID/beige mice (Taconic) mice were injected with 3 mg/kg sirolimus, 25 mg/kg PD0325901, or 3 mg/kg sirolimus + 25 mg/kg PD0325901 every 24 h for 3 d. Control mice were injected with a mixture containing ethanol and Cremophor EL in PBS. After the final Rapamycin treatment, mice were injected with 30 µg/kg recombinant human TNF (R&D Systems) diluted in 0.2 ml PBS via the tail vein. After 16 h, mice were anesthetized and perfused with saline, and portions of the aorta, kidney, liver, and heart were harvested. Tissue samples were then either flash frozen in liquid nitrogen or frozen in optimal cutting temperature (OCT) compound (Sakura). Serial 5- or 30-µm transverse sections were cut for immunofluorescence or quantitative real-time RT-PCR analysis, respectively.
Rapamycin
Manufactured by R&D Systems
Sourced in United States
Rapamycin is a macrolide compound that functions as an immunosuppressant and antiproliferative agent. It is a potent inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, which is involved in cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Sirolimus, by Taconic Biosciences (1 mentions)
Recombinant human tnf, by R&D Systems (1 mentions)
Cremophor el, by Merck Group (1 mentions)
Pd0325901, by Taconic Biosciences (1 mentions)
C b 17 scid beige mice, by Taconic Biosciences (1 mentions)
Pd0325901, by Selleck Chemicals (1 mentions)
Romidepsin, by Selleck Chemicals (1 mentions)
Ulixertinib, by Selleck Chemicals (1 mentions)
Otx015, by Cayman Chemical (1 mentions)
Gdc 0941, by Chemietek (1 mentions)
Azd8055, by Chemietek (1 mentions)
Nvp bez235, by Chemietek (1 mentions)
Q vd oph, by Apexbio (1 mentions)
Leupeptin, by Thermo Fisher Scientific (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Cxcl1, by Thermo Fisher Scientific (1 mentions)
Pepstatin a, by Bio-Techne (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
12 protocols using rapamycin
1
Rapamycin and MEK Inhibitor in SCID Mice
2
Combinatorial Inhibitor Treatment Protocol
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The HDI romidepsin (Cat #S3020) and the ERK inhibitor ulixertinib (Cat #S7854) were purchased from Selleck Chemicals (Houston, TX, USA). The PI3K inhibitor GDC-0941 (Cat #CT-G0941) and mTOR inhibitors AZD-8055 (Cat #CT-A8055) and NVP-BEZ235 (Cat #CT-BEZ) were from ChemieTek (Indianapolis, IN, USA). The mTOR inhibitor rapamycin (Cat #1292) was obtained from Tocris/R&D Systems (Minneapolis, MN, USA). The BRD inhibitor OTX-015 (Cat #15947) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). The pan-caspase inhibitor Q-VD-OPh (Cat #A1901) was obtained from ApexBio Technology (Houston, TX, USA).
3
Modulating IL-13-Induced Keratinocyte Response
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HaCaT cells, which are immortal human keratinocytes, were cultured in Dulbecco's modified eagle medium with 1.3 mmol/L calcium (PromoCell GmbH, Heidelberg, Germany) at 37 °C, 5% CO2. 24h prior to treatment, HaCaT cells were seeded at 2×105 cells/mL. Then, HaCaT cells were treated with 50 ng/mL of recombinant human IL-13 cytokine (R&D systems, Minneapolis, MN) for 48 h, except the blank control. 24 h after the stimulation, one group of HaCaT cells was treated with 20 ng/mL rapamycin (R&D systems), while another group with DMSO as the negative control.
4
Cytokine Effects on Human Motor Neurons
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hMNs were fluorescence-activated cell sorting (FACS) purified and plated on PDL/laminin-coated 384-well plates at a density of 5,000 cells/well. One day after cell plating, motor neurons were treated with 0.1% BSA in PBS or cytokines (TGF-β1, R&D Systems, IL-6, CXCL1, CXCL12, INF-ϒ, IL-1β, TNF-α, GDNF, BDNF, CNTF, LCN2, SERPINA3 [Peprotech]) at three concentrations, 2, 20, and 200 ng/mL. The media containing the various cytokines were changed every other day for 14 days. For adult conditioned medium (ACM) experiments, TGF-β1 inhibitor (RepSox; 8 μm) or mTOR inhibitor (rapamycin; 200 nM, R&D Systems) were added to ACM 1 day after plating FACS-purified hMNs. After 14 days of culture, motor neurons with various treatments were either fixed for analysis or used for western blot analysis. For autophagy flux analysis the cocktail of lysosomal inhibitors contained E-64-D (10 mg/mL, Enzo), pepstatin A (10 mg/mL, Tocris), and leupeptin (100 μM, Fisher).
5
Scratch Migration Assay for UC-MSCs
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The scratch migration assay was performed using 6‐well plates (Corning Costar, Corning, NY) at a density of 3 × 106 cells/well. When the cultured UC‐MSCs had reached 70%‐80% confluence, a line was scratched using a 1‐mL pipette tip. The cells were then randomly divided into six groups and treated with 3‐MA, rapamycin, rapamycin + AMD3100, a CXCR4 inhibitor (5 mg/mL; Cayman Chemical Company, Ann Arbor, MI), rapamycin + CXCL12, a CXCR4 agonist (50 ng/mL; R&D Systems, Minneapolis, MN), shCXCR4‐UC‐MSCs + rapamycin or common UC‐MSC medium. Each well was also treated with mitomycin C (10 µg/mL; Sigma‐Aldrich) to inhibit cell proliferation. Images were acquired at 0 and 24 hours under an inverted microscope (Leica, Mannheim, Germany) for cell counting.
6
Assessing Cell Proliferation with CFSE
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Fifty (50) million cells were stained with 5 μM CFSE (Life Technology, Grand Island, NY) for 5 min at room temperature. The cells were then washed twice with complete medium and added to 12-well plates. For proliferation analysis, the 1 x 105 CFSE-labeled cells were plated in 12-well plates alone, or in wells containing 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO), 1 mM Pirfenidone (R&D systems, Minneapolis, MN), 300 nM BIBF-1120 (Selleckchem.com, Houston, TX) or 1 nM Rapamycin (R&D systems, Minneapolis, MN) in the presence or absence of 2 x 105 fibroblasts for 72 h. The cells were then washed and fixed in 5% neutral buffered formalin (NBF) and analyzed by flow cytometry. Mean fluorescence intensities (MFI) were determined using FlowJo (TreeStar Inc.). The fold MFI change was determined relative to untreated cells and the change in proliferation was determined by taking the inverse of the fold change to reflect the inverse correlation between CFSE MFI and proliferation.
7
Effects of Pharmacological Modulators on Apoptosis
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5 x 105 of naïve CEM or fibroblast conditioned CEM cells were plated into cell culture dishes and treated with 1 nM Rapamycin (R&D systems, Minneapolis, MN), 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, MO), or 1 μM JAK1/2 inhibitor (A gift from Dr. Kojo Elenitoba-Johnson) for 24 hours. Cells were then fixed, and TUNEL analysis was performed using a TiterTACS Colorimetric Apoptosis Detection Kit as recommended by the manufacturer (Trevigen, Gaithersburg, MD).
8
Ovarian Tissue Culture with Rapamycin and NGF
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Ovaries from the P3 female pups were harvested and cultured on inserts (PICM01250, Millipore, Billerica, MA, USA) with 0.4 ml culture medium added to the bottom of each well. Each insert held 4–5 ovaries and a drop of medium was added to the top of each tissue fragment to prevent drying.17 (link) The culture medium was MEMalpha supplemented with 0.23 mM pyruvic acid, 50 mg/l streptomycin sulfate, 75 mg/l penicillin G and 3 mg/ml BSA. Ovaries were randomly distributed to the control and the treated groups. In the treated groups, the ovaries were incubated with 250 nM rapamycin (Sigma), 100 nM recombinant mouse beta-NGF (1156-NG/CF, R&D, Minneapolis, MN, USA) or rapamycin together with NGF for 24 h. After washing out the chemicals, the culture continued in control medium for 96 h (24+96 h) and ovaries were collected at 24 and 96 h, respectively.
9
Rapamycin-Induced IL15 Receptor Modulation
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The ESCs (2 × 10 5 cells/well) during the logarithmic phase were gently placed in 24-well flat-bottom plates and were treated with rapamycin (1 μM, R&D Systems) for 48 h. Then, FCM was applied to evaluate the IL15 receptor level on the ESCs.
10
Lipid Extraction and Autophagy Induction
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Unless otherwise specified, chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), culture media and supplements for cell culture from Gibco-Invitrogen (Carlsbad, CA, USA) and plasticware from Corning (Corning, NY, USA). Human osteosarcoma U2OS cells, their GFP-LC3-expressing derivatives, human neuroblastoma H4 GFP-LC3 (gift from Y. Juan) cells were cultured in DMEM medium containing 10% foetal bovine serum, 100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulphate (37 °C, 5% CO2). Lipid extractions were performed after 12 h of 10 mM ethanolamine (Sigma, E9508) treatment. For autophagy induction, cells were treated for 12 h with 10 mM ethanolamine or incubated in absence of nutrients. For the yeast-like chronological senescence assay, cells were treated for 1 week with 10 mM ethanolamine or 1 μM rapamycin (R&D Systems, Minneapolis, MN, USA).
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