HDAC inhibitory activity assay

The HDAC inhibitor drug screening kit (k340-100, BioVision, CA, USA) was used to measure HDAC inhibitory activities of the candidate compounds according to the manufacturer’s instructions. The candidate compounds, assay buffer, and HDAC fluorometric substrate, which comprises an acetylated lysine side chain, were added to HeLa nuclear extracts in a 96-well plate and incubated at 37 °C for 30 min. The reaction was stopped by adding lysine developer, and the mixture was incubated for another 30 min at 37 °C. An additional positive control included incubation with double-distilled water, and the inhibitor control consisted of incubation with Trichostatin A (TSA) at 20 μM. HDAC activities were quantified by a fluorescence plate reader (POLARstar OPTIMA, BMG, BRD) with excitation at 370 nm and emission at 450 nm.