Epoch colorimetric plate reader

HDAC activity was measured with 1.545 μg of nuclear protein extract following the manufacturers protocol (HDAC Assay Kit, colorimetric, Active Motif, Carlsbad, CA). As suggested in the protocol for samples with potentially low HDACs, we extended the initial HDAC reaction time to three hours. Since the kit was developed based on nuclear extracts from mammalian cells, we envisioned that extending the incubation time will help complete the deacetylation reaction. Samples were measured in triplicate (standards were measured in duplicate) using EPOCH colorimetric plate reader (Biotek, Winooski, VT) at 405 nm. In the assay reaction, a short peptide substrate was added along with the nuclear extract and other reagents as per the protocol. This substrate contains acetylated lysine residues and can be deacetylated by most HDAC enzymes. Active HDACs from the experimental samples would then bind to the added substrate by removing acetyl groups from the substrate. This reaction then yielded an HDAC-deacetylated colored product, which was measured by the colorimetric plate reader. The amount of deacetylated product in the reaction is directly proportional to the amount of active HDAC enzymes present in our samples [37 (link)].

To determine the concentration response curves for each of the drugs, JIMT-1 cells were passaged at confluency and the cells were seeded in 96-well plates at a seeding density of 5000 cells per 100 μL in each well. Once the cells were allowed to adhere for 36 h, they were treated with a range of PAC concentrations (0–5 μM), DAS concentrations (0–20 μM), and EVE concentrations (0–20 μM) in triplicate. The stock solutions were prepared in DMSO for all the drugs with final DMSO concentrations in the treated wells maintained below 0.1% and a vehicle control with 0.1% DMSO was included for each experiment. Following treatments, PAC-treated cells were incubated for 48 h and EVE- and DAS-treated cells for 72 h before measuring the percentage cell viability. After the treatment period, 10 μL of CC-8 reagent was added and incubated for 90 min. The absorbance of the wells was measured using an EPOCH colorimetric plate reader (BioTek, Winooski, VT, United States) at 450 nm. Relative absorbance of the treatment wells was normalized to the vehicle control to obtain percentage viability.