4.3. HDAC Enzyme Activity Assay

Pan HDAC enzyme (all the known class I HDACs-HDAC1, HDAC2, HDAC3, HDAC8 and class II HDACs 4-7, 9 and 10), HDAC subtype 1, 3 and 4 activity were determined by using the HDAC fluorometric cellular activity assay (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Different concentrations of AzP and SAHA were used, and the fluorescence intensity was measured on a fluorometric reader using excitation/emission wavelength of 360/460. Percent activity for each of the test compounds is calculated as follows:

Activity = {(Sc − B)/(S° − B)} × 100. Wherein, S c represents the signal measured in the presence of AzP or SAHA, S° denotes signal measured in the absence of AzP or SAHA, B is measured in blank wells containing the medium alone. IC₅₀ corresponds to the drug concentration which achieves 50% activity of the untreated control. IC₅₀ was then calculated and represented as mean ± SD.