Mgcd0103

To assess effect of short-term drug pretreatment on 2D/3D growth, cells are plated in six-well plates and allowed to recover for 24 h. At this point, drug-containing media was added to each well. After 24 h of treatment, cells were then were plated for mammospheres or 2D proliferation assays. 2D proliferation was determined after 3 days by viable cell counts, and assessed as a percentage of the DMSO control. All treatments were carried out in triplicate, and repeated a minimum of three times. The error bars represent the standard error of the mean of the three replicates. The small molecule drugs that are used to treat the cells for various experiments are: TSA (Selleck) Doxirubicin (Selleck), Paclitaxel (Selleck), 5-Fluorouracil (Sigma), SAHA (Selleck), GSK126 (Cayman Chem), MS275 (Selleck), MGCD0103 (Selleck), MC1568 (Selleck), MC1575 (Gift of Dr. Antonello Mai), Apicidin (Sigma), Droxinostat (Selleck). All drugs are prepared in DMSO, which was used as vehicle control in each assay.

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.

MGCD0103 and LBH589, were purchased from Selleck Chemicals, and trichostatin A (TSA) from Sigma Aldrich. The HDAC6 selective inhibitors Tubastatin A and Nexturastat were synthesized by Dr. Alan Kozikowski (University of Illinois, Chicago, IL). All HDACi were reconstituted in DMSO at greater than 1000× the final effective dose and stored in aliquots at −80 °C. For in vitro use, stocks were diluted in complete medium immediately before use. For in vivo studies, Nexturastat and Tubastatin A were dissolved in 10% DMSO plus 90% Hank’s buffered salt solution (HBSS) 1×.

LBH589 was kindly provided by Novartis (Basel, Switzerland). MGCD0103, MS275, PXD101, PCI34051, and ACY1215 were purchased from Selleck Chemicals (Houston, TX). DMSO-reconstituted HDACi aliquots were stored at −80°C. For in vitro studies, stocks were diluted to final concentration immediately prior to use. For in vivo use, LBH589 was dissolved and sonicated in 5% dextrose.

MGCD0103 and LBH589, were purchased from Selleck Chemicals. The HDAC6 selective inhibitors Tubastatin A and Nexturastat B were synthesized by Dr. Alan Kozikowski (University of Illinois, Chicago, IL). All HDACi were reconstituted in DMSO at greater than 10 mM and stored in aliquots at −80 °C. For in vitro use, stocks were diluted in complete medium immediately before use. For in vivo studies, Nexturastat B was dissolved in 5% DMSO plus 95% Hank's buffered salt solution (HBSS) 1X.