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In vitro fluorescent histone deacetylase assay: HDAC inhibition assays were performed using the HDAC fluorescent activity assay kit (Biomol Research Laboratories, Plymouth Meeting, PA). HeLa cell nuclear extract, which contains a number of HDAC isozymes and other nuclear factors, was used as the source of HDAC activity. The final substrate concentration in the assay mixture was 50 μM. The reaction was allowed to proceed for 10 min at room temperature before a stop solution was added. Test compounds were prepared as 20 mM stock solutions in DMSO (Molecular Biology grade, Sigma-Aldrich Co., St. Louis, MO) and stored at -70 °C. DMSO had no significant effect on the activity of this assay at concentrations up to 5% with the final DMSO concentration in the assays of no more than 2%. Assays were performed in white polystyrene 96-well half-area assay plates (Corning, Corning, NY) and measured on a Wallac 1420 fluorescent plate reader (Wallac Oy, Turku, Finland) with an excitation wavelength of 355 nm, an emission wavelength of 460 nm, and a one sec signal averaging time.
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