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. Afterwards, 125 ul of propidium iodide was added to each sample and was kept at 4 C in dark before flow cytometry. check FAQ Wound healing, cell migration, and invasion assays The wound healing assay was performed as follows. Equal numbers of cells were cultured in full medium in a 6 well plate until 90% confluency. Cells were then pretreated with 10 ugml of mitomycin C for 2 h, and Inhibitors,Modulators,Libraries three parallel wounds were created in each plate with a sterile 200 ul pipette tip. The plate was then washed with PBS, and the width of the wounds was photographed at differ ent time points. The relative vel ocity of cell migration was calculated as the change in widthtime.
Quantification of cell migration and invasion was Inhibitors,Modulators,Libraries per formed using QCM 24 Well Colorimetric Cell Migration and Cell Invasion Assay Kits. Briefly, cells were resuspended in serum free culture medium and then seeded on the upper chamber. The full medium was then placed in the lower chamber as a chemo attractant, and the cells were allowed to pass through the pores to the lower surface of the membrane. The cells were then stained with the staining buffer and photo graphed in three different microscopic fields. Statistical analysis The SPSS 14. 0 software was used for statistical analysis. Fishers exact test and the Mann Whitney test were used to compare the values between sub groups, and data were expressed as the Inhibitors,Modulators,Libraries mean SD.
The Students t test was used to compare the values between subgroups, and P 0. 05 was considered to be a statistically significant difference between groups of data. Results Reduced expression of AMPK B1 during ovarian cancer progression AMPK B1 expression in clinical samples was analyzed using immunofluorescence and IHC analyses. We first ex amined the subcellular localization Inhibitors,Modulators,Libraries of Inhibitors,Modulators,Libraries AMPK B1 in ovarian cancer cells. Using an immunofluorescence analysis, we observed Sorafenib Raf-1 an accumulation of GFPAMPK B1 at the plasma membrane and as punctate structures throughout the cytoplasm of SKOV3 cells. However, our previous qPCR analysis showed that the expression of AMPK B1 was significantly reduced in late stage compared to early stage ovarian cancer. Similarly, our current analysis using IHC also showed that the AMPK B1 level was reduced in early to advanced stage ovarian cancers. The reduced AMPK B1 level was signifi cantly associated with late stage, high grade and metastatic ovarian cancers. More importantly, we observed that the expression level of AMPK B1 exhibited a stepwise re duction pattern that accompanied the tumor stage pro gression of ovarian cancers.
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