Lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 4% to 20% gradient gels (Pierce/Thermo Fisher Scientific, Waltham, MA). Separated proteins were electrophoretically transferred to Immobilon P membranes (Millipore, Bedford, MA) and probed with specific phospho-p44/42 mitogen-activated protein kinase (MAPK; Thr202/Tyr204; Cell Signaling, Beverly, MA) and total extracellular Alectinib order signal-regulated kinase (ERK)1/2 (Santa Cruz Biotech, Santa Cruz, CA) antibodies. Cell proliferation

was determined using a colorimetric assay, CellTiter 96 AQueousOne Solution Cell Proliferation Assay (Promega, Madison, WI). Cells were plated in triplicate in 96-well plates (2.5 × 103 cells/well). Twenty-four hours later, PD0325901 or vehicle

control (dimethylsulfoxide) was administered. After treatment for the indicated time, cells were incubated with 20 μL CellTiter 96 AQueous One Solution Reagent, and absorbance was recorded at 490 nm. Percent cell growth was determined from a ratio of average absorbances of the treatment wells to the control wells. HepG2 or Hep3B cells were plated in 96-well plates. The next day, PD0325901 or vehicle was added for 48 hours. Relative apoptosis was determined using the Cell Death Detection enzyme-linked immunosorbent assay (Roche, Indianapolis, IN) by comparing the absorbance of drug-treated with that of vehicle-treated cells. Six-week old athymic mice (Harlan, Indianapolis, IN) were obtained, and 1 × 107 TAMH cells were injected into the right flank. The tumors were allowed to grow for 5 weeks before the start of treatment. The first arm was treated for 24 hours, and the second

see more arm was treated for 16 days. The first arm of this trial consisted of 10 mice divided into two groups, receiving either a single dose of PD0325901 (20 mg/kg) or an equivalent volume of vehicle (0.5% hydroxypropyl methyl cellulose, 0.2% Tween 80 [HPMT]) via orogastric gavage. After 24 hours, the animals were sacrificed, and the flank tumors were excised, frozen in liquid nitrogen, and stored at −80°C for ex vivo tumor analysis. The second arm of this trial consisted of 17 athymic mice receiving either PD0325901 or HPMT vehicle Carnitine palmitoyltransferase II daily by orogastric gavage. Tumor volume was measured twice per week via vernier caliper (Scienceware, Pequannock, NJ). Volume estimations were determined by the following formula: Hep3B cells (1 × 106 cells) were injected into the flanks of athymic mice. When tumors were visible, treatment with either PD0325901 (10 mg/kg) or vehicle was initiated and tumor volume monitored as described for 4 weeks. All animals were housed and fed in American Association for Accreditation for Laboratory Animal Care–approved facilities, and animal research and handling were in strict conformance with federal Institutional Animal Care and Use Committee guidelines. MT42 (CD-1) TGF-α transgenic mice (provided by Glen Moreno) were injected with diethylnitrosamine (5 mg/kg) at 14 days of age.