Cudc 101

Cells were plated in DMEM with 2% FBS in 96 well plates at 3,000 cells per well. 24 hours post-plating, cells were treated with a 9-point dose dilution of the indicated inhibitor, either as a single agent or in combination, with DMSO as a control. After 72 hours, viability was measured using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96 cell viability kit (Promega). Combination Indices (CI) were calculated using the Chou and Talalay method (24 (link)) with CalcuSyn software (CalcuSyn). Details of the cytotoxicity assays can be found in the Supplemental Methods. The therapeutic compounds used were as follows: OP449 (25 (link)) and DT1154 (16 (link)) were described previously. BEZ235, INK128, GDC-0068, PP242, CUDC101, VX680, XL880, and Dasatinib were purchased from Selleck Chem.

HDAC inhibitors (HDACi) CUDC-101 [19 (link)], Pracinostat [36 (link)], MGCD0103 [37 (link)], MC1568 [38 (link)] and PCI-34051 [39 (link)] were purchased from Selleck Chemicals. The specific inhibitors of EGFR and HER2/NEU, Gefitinib [40 (link)] and CP-724714 [41 (link)], were also purchased from Selleck. Sodium Butyrate [42 (link)] was purchased from Sigma Aldrich. All HDACi were used at published IC₅₀. Gefitinib and CP-724714 were used at a range of concentrations (108-540 nM and 108-864 nM, respectively). In the experiments determining IC₅₀, dose response and time course activity against AR-V7 and flAR activity (Fig. 1), CUDC-101 was used between 0.3 nM to 1 uM from 3 to 24 hours. It was used at a concentration of 300 nM for 6 or 24 hours in the remaining experiments, unless stated otherwise. All reagents were dissolved in DMSO, except DHT, which was dissolved in ethanol.