YM201636 (IC50=33 nM), a specific PIKfyve inhibitor, is less effective to p110α and insensitive to Fabl, which is yeast orthologue.

PIKfyve:33 nM

In NIH3T3 cells treated with serum starvation, YM201636 (0.8 μM) significantly reduces the production of PtdIns(3,5)P2 by up to 80%. In 3T3-L1 adipocytes, YM-201636 (IC50=54 nM) inhibits the absorption of 2-deoxyglucose, and at a dose of 160 nM, it fully inhibits the absorption of 2-deoxyglucose. In MDCK cells, YM201636 disrupts the continuous recycling of the intercellular junction proteins Claudin-1 and Claudin-2, leading to their accumulation within the cells and delaying the formation of epithelial barriers.

Following 3T3L1 adipocyte serum-starvation and insulin stimulation, cell lysates containing protease inhibitors are clarified and then subjected to immunoprecipitation with anti-PIKfyve antibodies. Washed beads are mixed with 100 μM PtdIns and preincubated for 15 min with YM-201636 (100 nM) or vehicle in the assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA and 10 mM MgCl2). The kinase assay (50 μL final volume) is carried out for 15 min at 37 °C with 15 μM ATP and [γ-32P]ATP (30 μCi). Lipids are extracted, spotted on TLC glass plates (250 μm), resolved by a chloroform/methanol/water/ammonia solvent system and detected by autoradiography[2].

YM-201636 is dissolved in DMSO and diluted with DMEM and added to cells at a final concentration of 800 nM. Cells are treated with YM-201636 or a DMSO control for 2 h. For TER measurements cells are plated at confluency on Transwell permeable polyester filters (0.4 μM pore size) with surface area of 0.33 cm2. Media is changed ever 2-3 days and cells are grown for 7 days prior to TER measurements[4].