Cyclosporin A is a naturally occurring cyclic polypeptide that is the active metabolite of a fungus. Cyclosporin A is an immunosuppressant that binds to procyclins and inhibits calcineurin (IC50=7 nM).

Calcineurin phosphatase:7 nM

METHODS: Glioma cell C6 was treated with Cyclosporin A (10 μM) under hypoxic conditions for 4 h. The expression levels of target proteins were measured by Western Blot.
RESULTS: 4 h hypoxia induced a large accumulation of endogenous HIF-1α protein, and Cyclosporin A prevented most of the hypoxia-induced HIF-1α stabilization. [1]
METHODS: Human colon cancer cells CACO-2 were treated with Cyclosporin A (2 μM) for 24-72 h. The cell cycle was detected by Flow Cytometry.
RESULTS: Accumulation of cells was detected in the G0/G1 phase after treatment with Cyclosporin A. The RESULTS were summarized in the following table. [2]

METHODS: To assay activity against muscle disease in vivo, Cyclosporin A (5 mg/kg in olive oil) was injected intraperitoneally into myopathic Col6a1-/- mice twice daily for ten days.
RESULTS: Cyclosporin A affected satellite cell activity and triggered regenerative fiber formation in Col6a1-/- mice. [3]
METHODS: To detect the effects on ischemia-reperfusion injury (IRI), Cyclosporin A (3 mg/kg, 1 h or 10 min before ischemia; 10 mg/kg, 10 min before ischemia) was intraperitoneally injected into ischemia-reperfused C57BL/6J mice.
RESULTS: Mortality and renal function were significantly higher in the Cyclosporin A 10 mg/kg-10 min and Cyclosporin A 3 mg/kg-1 h groups than in the Cyclosporin A 3 mg/kg-10 min group. [4]

Phosphatase Assay: Purified bovine brain calcineurin and calmodulin are purchased. Reaction mixtures with purified enzyme contains 100 nM calcineurin, 100 nM calmodulin, and 5 μM 32P-labeled phosphopeptide, in 60 μl (total volume) of assay buffer containing 20 mM Tris (pH 8), 100 mM NaCI, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mg of bovine serum albumin per ml, and either 0.1 mM CaCl2 or 5 mM EGTA. Reaction mixtures with cell lysates contains 20 μl of undiluted lysate, 5 μM 32P-labeled phosphopeptide, and 40 μl of assay buffer. Where indicated, reaction mixtures contains 50 μM peptide 412 or 413 and/or 500 nM okadaic acid, a specific inhibitor of phosphatases 1 and 2A; 500 nM okadaic acid is sufficient for inhibition of Ca2+-independent phosphatases, whereas higher concentrations partially inhibit Ca2+-dependent activity as well. After 15 min at 30°C, reactions are terminated by the addition of 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 5% trichloroacetic acid. Free inorganic phosphate is isolated by Dowex cation-exchange chromatography and quantitated by scintillation counting as described.

Immunosuppressive agents are dissolved in ethanol at concentrations 1000-fold more than the concentration desired for cell treatments. Cells (106) are suspended in 1 ml of complete medium in microcentrifuge tubes; 1 μl of ethanol or of the ethanolic solution of Cyclosporin A is added, and the cells are incubated at 37°C for 1 hr. Cells are washed twice with 1 ml of PBS on ice and lysed in 50μl of hypotonic buffer containing 50 mM Tris (pH 7.5); 0.1 mM EGTA; 1 mM EDTA; 0.5 mM dithiothreitol; and 50 μg of phenylmethylsulfonyl fluoride, 50 μg of soybean trypsin inhibitor, 5 μg of leupeptin, and 5 μg of aprotinin per ml. Lysates are subjected to three cycles of freezing in liquid nitrogen followed by thawing at 30°C and then are centrifuged at 4°C for 10 min at 12,000×g.(Only for Reference)