Histone Deacetylase Activity Assay

CEFs were treated with HDACi compounds for 48 h, as described earlier. Nuclei were isolated and extracted from 1 × 10⁷ cells according to the manufacturer's instructions (Histone Deacetylase Activity kit, Abcam, ab156064) (30). Briefly, cells were resuspended in 1 ml of lysis buffer followed by centrifugation over a 30% sucrose solution. Nuclei pellets were washed and resuspended in an extraction buffer. Samples were sonicated for 30 s (EpiShear Q120AM probe sonicator, Active Motif) followed by a 30-min incubation on ice. The supernatants (i.e., crude nuclear extracts) were flash-frozen in LN₂ and stored at −80°C until further use. Small aliquots of the supernatants were used to determine protein concentration by the Bradford method using protein dye concentrate. All experiments were carried out according to the manufacturer's instructions, following the two-step method and using the controls provided by the kit. The fluorescence intensity was measured in a microplate fluorescence reader (Molecular Devices FilterMax F3) at Excitation/Emission = 350–380/440–460 nm.