Histone deacetylase hdac activity assay kit fluorometric

RD (ERMS) and RH30 (ARMS) human cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were maintained as already described [80 (link),81 (link)]. RD and RH30 were cultured respectively Dulbecco’s Modified Eagle’s and RPMI medium containing 10% Fetal Calf Serum (Hyclone, Logan, UT, USA) supplemented with glutamine and gentamycin (GIBCO-BRL Gaithersburg, MD, USA). GenePrint 10 System (Promega Corporation, Madison, WI, USA) was used to authenticate cell cultures by comparing the DNA profile of our cell cultures with those found in GenBank. The cells were dissociated using 0.25% trypsin and 0.02% EDTA solution and resuspended into a fresh medium once every 2–3 days. For the experiments, cells were seeded into 6-well tissue culture plates at a density of 8500 cells/cm². One day after plating, cells were treated with Entinostat (MS-275) purchased from Selleckchem.com (Houston, TX, USA). Trypan blue (Thermofisher) exclusion was used to assess cell viability. Countess II Automated Cell Counter (ThermoFisher Scientific, Waltham, MA, USA) was used to assess the number of the cells. SigmaPlot (Systat Software, Inc, San Jose, CA, USA) software was used to calculate IC₅₀ values. Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric) (ab156064) from abcam was used to test HDACs activity.

Prostate cancer cell lines, AR− (PC3 and DU145 cells) and AR+ (LnCAP cells) and mouse embryonic fibroblasts (NIH 3T3 cells) were obtained from American Type Culture Collection (Manassas, VA, USA). The epithelial prostate cell line immortalized with SV40 (PNT1A) was gifted from the Department of Anatomy, University of Otago. Primary antibodies to acetyl H3, cyclin D, cyclin B, p21, p27kip1, and bcl-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). β-tubulin, β-actin, Dulbecco’s modified Eagle’s medium (DMEM) nutrient mixture Ham’s F-12, sulforhodamine B salt and propidium iodide (PI) were purchased from Sigma-Aldrich (Auckland, New Zealand). Acrylamide, bisAcrylamide, sodium dodecylsulfate and PVDF membrane were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Complete mini-EDTA-free protease inhibitor was purchased from Roche Diagnostics Corporation (Mannheim, Germany). Annexin V APC was obtained from BD Pharmingen (San Jose, CA, USA). FxCycle PI RNase staining solutions were ordered from Life Technologies (Christchurch, New Zealand). The Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric) was purchased from Abcam (Melbourne, Australia). Jazz90 and Jazz167 were synthesized as previously described [23 (link)]. SAHA was obtained from AK Scientific (Union City, CA, USA).