Ms 275

Libraries used for high-throughput drug screening are described in Figure 1. Other compounds used include the HDAC inhibitors SAHA, HNHA, LBH-589, Scriptaid, MS-275, Givinostat, PDX101, LAQ-824 and MGCD0103 (all from Selleck), the PP2A inhibitors Cantharidin and NorCantharidin, the topoisomerase inhibitors Camptothecin and Topotecan (all from Sigma), the HMG CoA reductase inhibitors Cerivastatin (Sigma) and Itavastatin (Sequoia Research Products Ltd. UK), and the PI3K/mTOR inhibitors BEZ-235 and BKM-120 (both from Active Biochem). All inhibitors were dissolved in DMSO to 20 mM, and further diluted to an appropriate final concentration in culture medium at the time of use.

Immortalized mouse proximal tubule cells(TKPTS) [32 ], kindly provided by Dr. Bell-Reuss and Dr. J. Megyesi, were cultured in DMEM/F12 supplemented with glutamine, 7.5% FBS and antibiotics and were grown to confluence and maintained at 37° C in 5% CO₂. Cells were treated with cisplatin with/without 1μg/ml of AMWAP or 0.1μM of TSA for 24 h and then harvested for RNA isolation or used to determine apoptosis. To determine the specific HDAC that regulates AMWAP expression, cells were treated with vehicle or specific inhibitor for HDAC-1 (MS-275, 1μM), HDAC-1&2 (romidepsin or FK228, 200nM) or HDAC-6 (Tubastatin A, 100nM) (SelleckChem, Houston TX, USA) for 24hrs. Cells were harvested and RNA used for AMWAP expression studies by real-time PCR. To determine the effect of AMWAP knockdown on TSA-induced suppression of cisplatin-mediated epithelial cell apoptosis, siRNA specific to AMWAP was transfected (50nM). 48hr after transfection, cells were treated with/without cisplatin and TSA for 24hr, and then cells were harvested to quantify apoptosis by flow cytometry.
Raw 264.7 cells were cultured in RPMI medium containing 10% FBS. At 80% confluency, cells were treated with cisplatin with/without 1μg/ml of AMWAP or 0.1μM of TSA for 24 h and 72hrs. Cells and supernatants were harvested and subjected to cytokine and gene expression analysis.

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.