The blots were then incubated with goat anti rabbit or anti mouse secondary antibodies that were conjugated to horseradish pero idase and visualized via an enhanced chemiluminescence system. B selleckchem Actin was used as the loading control. For immunofluorescence analysis, SKOV3 cells were cultured on cover slips and transiently transfected with AMPK B1 e pressing plasmid. The preparation and e am ination of pEGFP AMPK B1 transfected cells were per formed as previously described. Immunohistochemical staining for AMPK B1 was performed on an ovarian cancer tissue array, and an antibody against AMPK B1 was used to e amine the e pression of AM PK B1. Procedures and the scoring of results were per formed as previously described, and the e amin ation of immunohistochemical staining was performed by two independent observers.

Confocal microscopy The cellular localization of AMPK B1 was e amined in A2780CP and SKOV3 cells after the transient e pression of the pCMV6 AMPK B1 GFP tagged plasmid. The analytical procedure was reported pre viously, and fluorescence signals were captured using confocal microscopy. Cell proliferation assay The cell proliferation assay was performed using a cell proliferation kit, and data were obtained from three separate e periments that were performed in triplicate. Clonogenic assay Appro imately 800 cells were plated in triplicate in 6 well plates to form colonies for up to 2 4 weeks, and the medium was replaced every 3 7 days. The colonies were then stained with crystal violet and counted.

Anchorage independent growth assay A soft agar colony formation assay was used to determine the capacity of ovarian cancer cells to undergo anchorage independent cell growth upon different treatments. Sterile 2% and 0. 6% agarose gel stocks in 2�� MEM containing 20% FBS were prepared, and single cell suspen sions were prepared by suspending 1000 cells in 2 ml of full medium containing 0. 3% agar. The cell suspensions were plated on top of a solidified bottom layer with 1% agar in the full medium, and the plates were incubated at 37 C in a humidified incubator for 14 21 days. The col onies were then counted using a dissecting microscope. Flow cytometry The DNA content, cell cycle distribution and percentage of apoptotic cells of each sample were assessed by flow cytometry. Cells were cultured Brefeldin_A in 6 well plates, and float ing and attached cells were harvested by trypsinization, centrifuged and resuspended in PBS. The cells were then fi ed overnight with 1 ml of 70% ethanol at 4 C followed by centrifugation at 4,000 g at 4 C for 5 min and one wash with ice cold PBS. RNase A was heated at 95 C for 10 minutes before use, and the cell pellets were resus pended in 500 ul of PBS containing 5 ul of RNase A and then incubated at 37 C for 30 min.