| In Vitro | In vitro activity: In LNCaP prostate cancer cells, CID755673 directly inhibits PKD1 activity. In HeLa cells, CID755673 significantly blocks PMA-induced nuclear export of HDAC5, and blocks PKD-mediated protein transport. In prostate cancer cells, CID755673 potently blocks cell migration and invasion, and also shows inhibition activity on tumor cell proliferation and cell cycle distribution. In addition, CID755673 alter primary human NK cell effector functions.
Kinase Assay: The radiometric kinase assay is carried out by coincubating 0.5 μCi of [γ-32P]ATP, 20 μM ATP, 50 ng of purified recombinant human PKD (PKD1, PKD2, and PKD3) or CAMKIIα proteins, and 2.5 μg of Syntide-2 in 50 μl of kinase buffer that contains 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 10 mM β-mercaptoethanol. The reaction is carried out under conditions that the initial rate is within the linear kinetic range. The filter papers are then washed three times in 0.5% phosphoric acid, air-dried, and counted using a Beckman LS6500 multipurpose scintillation counter.
Cell Assay: Cell proliferation was determined by counting the number of viable cells (LNCaP or PC3 cells) upon trypan blue staining. Cell proliferation is measured by CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer's instructions. |
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