HDAC Inhibition Assay Protocol

The assays were carried out by Shanghai ChemPartner Co., Ltd (Shanghai, China), according to our previous method [23 (link),24 (link)]. Briefly, different concentrations of compounds were incubated with recombinant human HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8 (BPS Biosciences, San Diego, CA, USA) at room temperature for 15 min, which was followed by adding Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Reaction mixtures were incubated at room temperature for 60 min in HDAC1, HDAC2, HDAC3 and HDAC6 assays, and were incubated for 240 min in HDAC8 assay. Then, the stop solution containing trypsin was added. The coupled reaction was incubated for another 90 min at 37 °C. Fluorescent AMC released from substrate was measured in SynergyMx (BioTek, Winooski, VT, US) using filter sets as excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated by GraphPad Prism software (7.0 version., GraphPad Software, San Diego, CA, USA).