PCI-34051 is an effective and selective HDAC8 inhibitor (IC50: 10 nM).

HDAC1:4 μM, HDAC10:13 μM, HDAC6:2.9 μM, HDAC8:10 nM

Histone deacetylase activity:For PCI-34051 characterization, measurements are performed in a reaction volume of 100 μL using 96-well assay plates in a fluorescence plate reader. For each isozyme. The HDAC protein in reaction buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 5% dimethyl sulfoxide, pH7.4, supplemented with bovine serum albumin at concentrations of 0-0.05%) is mixed with PCI-34051 at various concentrations and allowed to incubate for 15 min. Trypsin is added to a final concentration of 50 nM, and acetyl-Gly-Ala-(N-acetyl-Lys)-amino-4-methyl coumarin is added to a final concentration of 25-100 μM to initiate the reaction. After a 30 min lag time, the fluorescence is measured over a 30 min time frame using an excitation wavelength of 335 nm and a detection wavelength of 460 nm. The increase in fluorescence with time is used as the measure of the reaction rate.

Cell lines: A549 cell line,Ovcar-3 cell line. Concentrations: 5 μM. Incubation Time: 24 hours. Method: Tumor cell lines and human umbilical vein endothelial cells are cultured for at least two doubling times,and growth is monitored at the end of PCI-34051 exposure using an Alamar Blue fluorometric cell proliferation assay as recommended by the manufacturer.PCI-34051 is assayed in triplicate wells in 96-well plates.The concentration required to inhibit cell growth by 50% (GI50) and 95% confidence intervals are estimated from the nonlinear regression using a four-parameter logistic equation.