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Materials and methods
HeLa, DU-145, A549, MiaPaCa2, and NCI-H460 were purchased from ATCC (Manassas, VA). All above cell lines were supplemented with 10% fetal bovine serum (FBS; v/v, Invitrogen) and 10 mM l-glutamine (Invitrogen). Human umbilical vein endothelial Q-VD-Oph (HuVEC), human primary mammary epithelial cells (HMEC) and human primary prostate epithelial cells (PrEC) were purchased from Lonza (Walkersville, MD).
Western blot analysis. HeLa cells or NCI-H460 cells were seeded at a density of 2.5 × 105 in 6-well plates with MEM plus 10% FBS for overnight. Cells were treated with various concentrations of AC-93253 for 16 h in the absence or presence of 40 nM TSA After 16 h, cells were lysed in RIPA buffer (Sigma) containing protease inhibitor cocktail (Roche, Palo Alto, CA) and phosphatase inhibitors (Sigma). Total cell lysates were denatured and resolved on SDS–polyacrylamide gels, and transferred onto nitrocellulose membranes. Western blot was performed with 1:1000 dilution of primary antibodies and 1:10000 dilution of secondary antibodies. Image scanning and quantification was performed with Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE) according to manufacturers’ instructions.