1% DMSO. After 24, 48, and 72 h, cell survival and selleck compound growth were measured by the Cell Titer 96 Aqueous MTS Reagent (Promega, Madison, WI) according to the manufacturer’s protocol. All experiments were performed in triplicate and repeated three times, independently. The light absorbance was measured by using an automatic microplate reader (Epoch, Bio-Tek Instruments, Winooski, VT) at 490 nm (14). Data were expressed as a percentage versus control (vehicle set at 100%). HCT-116 and SW-480 cells were seeded in 24-well plates. After 24 h, the medium was changed
and PPD was added at different concentrations. After treatment for 48 h, all adherent cells were collected with 0.05% trypsin, including the floating cells in the medium, and centrifuged for 5 min at 600 g. Then, the cells were double stained with Selleckchem NVP-AUY922 Annexin-V-(FITC) and propidium iodide (PI) (Becton Dickinson, San Diego, CA) according to the manufacturer’s instructions (15). Untreated cells were used as control. The stained
cells were subsequently analyzed by a FACS Canto flow cytometer (Becton Dickinson, Mountain View, CA). All experiments were performed independently three times, and run in triplicate. At least 10,000 cells were counted each time. Data were analyzed by FlowJo software 9.0. For cell cycle assay, 1 × 105 cells were seeded in 12-well plates. On the second day, PPD or vehicle was added. 48 h later, all adherent cells were collected by trypsin, fixed with 80% ethanol and stored for 2 h at −20 °C. After treatment with 0.25% Triton X-100 for 5 min, the cells were resuspended in 200 μL of PI/RNase staining buffer (Becton Dickinson, San Diego, CA), incubated in the dark for 20 min at room temperature,
and counted with a FACS Canto flow cytometer. At least PAK6 10,000 cells were counted for each measurement. Data were analyzed by FlowJo software 9.0. HCT-116 cells were plated at a density of 1 × 105 cells/dish in 60 mm tissue culture plates. Cells were allowed to adhere for 24 h before treatment. Thereafter, cells were treated with 20 or 25 μM of PPD for 24 or 48 h. Total RNA was extracted using miRNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instruction and quantified by NanoDrop (Thermo, Wilmington, DE) before hybridization. A group of 6 samples obtained from the in vitro assays were included in the cDNA array assays. Gene arrays were performed by using Affymetrix GeneChip Human Gene 1.0 ST Array (Dumbarton Circle, Fremont, CA), which contains 28,853 mouse genes being represented on the array by approximately 27 probes spread across the full length of a given gene. This provides a more complete and more accurate picture of gene expression than 3′-based expression array designs.