Five microliters of the reaction was treated with DpnI for 1 hour at 37°C prior to transformation. All constructs were verified by sequencing. Cells were cotransfected with 2 μg DNMT1-WT or DNMT1-MUT construct and 2 μg pRL-TK Renilla luciferase expression construct followed by a precursor miRNA at 100 nM final concentration selleck products using TransIT-LT1 and TransIT-TKO reagents (Mirus, Madison, WI) for DNA vectors and precursor miRNA, respectively. Luciferase assays were performed after 48 hours using the Dual Glo Assay system (Promega, Madison, WI) and a multiwell plate luminometer (Veritas, Turner Biosystems, Sunnyvale, CA). Cells grown in 100-mm culture dishes were washed twice with ice-cold phosphate-buffered

saline and then lysed by incubation for 20 minutes in 1 mL of ice-cold cell lysis buffer (Cell Signaling Inc., Beverly, MA). For analysis of xenograft tumor tissue, the tissue was homogenized, and lysates were obtained. The protein concentration in the lysates was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA). Equivalent amounts of protein were mixed with 4× sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer, electrophoresed in a 4% to 12% linear gradient Tris-HCl–ready gel (Bio-Rad), Alectinib in vitro and transferred to nitrocellulose membranes. The membranes were blocked

with 5% nonfat dry milk in Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 and were incubated with primary antibodies and IRDye700 and IRDye800-labeled secondary antibodies (Rockland, Gilbertsville, PA). The protein of interest was visualized and quantitated using the LI-COR Odyssey Infrared Imaging System (LI-COR Fossariinae Bioscience, Lincoln, NE). Eight-week-old male athymic nu/nu mice were obtained from Charles River Laboratories (Wilmington,

MA) and fed food and water ad libitum. The mice were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. They were housed three or four per cage, and fluorescent light was controlled to provide alternate light and dark cycles of 12 hours each. Mz-IL-6 or Mz-1 control cells (5 × 106 cells) were suspended in 0.25 mL of extracellular matrix gel, and the mixture was injected subcutaneously into the right and left flanks. Xenograft growth was monitored by serial measurements. For analysis of protein expression in vivo, the xenografts were excised once visible tumors had formed, and tissue was homogenized. An aliquot of the lysates was used for protein expression studies. Pre-miR miRNA precursors of pre-miR-148a, pre-miR-152, pre-miR-301 and control pre-miR-precursor were purchased from Ambion (Austin, TX). Antibodies against DNMT-1 and p16INK4a were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Rassf1A was obtained from Abcam (Cambridge, MA), and α-tubulin was obtained from Sigma (St. Louis, MO).