Hdac3

Four cell lines (human colon cancer cell line HCT116, human embryonic kidney cell line HEK293, human liver cancer cell line HepG2, and human B lymphoma cell line SU-DHL-6), fetal bovine serum (FBS), Eagle's Minimum Essential Medium (EMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from American Type Cell Collection (ATCC, Manassas, VA, USA). H9-derived human neural stem cells, defined FBS, trypsin-EDTA, fibronectin, basal fibroblast growth factor (bFGF), epidermal growth factor (EGF), KnockOut™ Dulbecco's Modified Eagle Medium (DMEM)/F12, and penicillin-streptomycin, StemPro^® Neural Supplement were acquired from Life Technologies, Carlsbad, CA, USA. Chemicals and epigenetic compound libraries were purchased from Cayman Chemicals (Ann Arbor, MI), Santa Cruz Biotechnology (Dallas, TX, USA), SelleckChem (Houston, TX, USA), and Sigma-Aldrich (St. Louise, MO, USA). HDAC-Glo I/II and CellTiter-Glo reagents were purchased from Promega, Madison, WI. Fluorogenic assay kits for HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10 were acquired from BPS Bioscience (San Diego, CA). White solid bottom 1536-well assay plates were purchased from Greiner Bio-One (Monroe, NC).

Human recombinant HDAC1, HDAC2, HDAC3 (BPS Bioscience) and HDAC8 (In-house purified from E-coli) were diluted with assay buffer 1 (25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂, 1 mg/mL BSA) to give 4, 5, 1, and 8.5 ng/µL stocks of each isoform, respectively. Serial dilutions of the compounds/PRPs were made in assay buffer 2 (25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂) starting with the highest concentration at 1 mM. 10 µL of the enzyme stock and 30 µL of each of the serial dilutions were mixed in a black half-area, low protein binding 96 well plate (Corning) and pre-incubated for 5 minutes (3 hours for the o-aminoanilide derivatives, 6, 13 and 14) at room temperature (rt). Next, 10 µL of 125 µM Boc-L-Lys(Ac)-AMC (BLA) fluorescent substrate (Chem-Implex) for HDACs 1–3 or 10 µL of 25 µM Fluor de Lys®, BML-KI178 (Enzo Life Sciences) for HDAC8 was added to each well and incubated for 30 minutes at rt. The reaction was quenched by the addition of 50 µL of 1 mg/mL trypsin and 5 µM TSA in assay buffer 2 and incubated for an additional 30 minutes. The fluorescence signal was read at excitation wavelength 360 nm and emission wavelength 460 nm using a Synergy 4 hybrid microplate reader from BioTek. The statistical data analysis and IC₅₀ values were determined using GraphPad Prism 7.02.