increased amounts of uPA and PAI 1 are generally linked with a far more extreme phenotype of ovarian cancer and are related to a poor prognosis. Confluent SKOV 3 monolayers were scratched with a yellow pipette tip, washed and then treated with serum free media containing the given levels of signal pathway inhibitors and/or blocking antibodies. Wound caused migration assays with Akt siRNA or Akt adenovirus treated SKOV 3 cells were done in 1000 FBS containing media. Extra injury caused migration assays with insulin and IGF 1 were performed on serum deprived ATP-competitive c-Met inhibitor SKOV 3 monolayers, as described previously. Cells were pre treated for 1 h with clean DMSO or 50 uMLY294002 in serum free media, damaged, washed and then treated with 10-0 nM insulin or 50 ng/mL IGF 1 in the absence or presence of 50 uM LY294002 in serum free media for 2-4 h. Migration of cells to the wound was administered, beginning at 0 h utilizing a MDS290 camera and software. Wound closure was quantified by measuring the distance in pixels between wound edges at every time point, using the measuring instrument in Adobe Photoshop with a superimposed on the image to steer the measurements. The wound size was normalized to one hundred thousand at 0 h for each treatment condition and presented as a per cent of wound remaining at the given time point. SKOV 3 cells were scratched with a sterile yellow pipette tip, plated on glass-bottom recipes over-night and treated with indicated concentrations Plastid of cell signaling inhibitors for 6 h. Low permeabilized cells were fixed in 1000 paraformaldehyde, then incubated with anti-bodies to PAI 1 and uPA. AlexaFluor conjugated secondary antibodies were used to imagine immunofluorescence staining using an Olympus FV500 confocal laser scanning microscope. An indirect uPA activity assay was performed using a synthetic plasmin substrate testing plasminogen activation by uPA as described previously. Shortly, unconcentrated conditioned media from treated cells was included with buffer containing plasminogen and plasmin substrate. Triplicate samples were incubated for 60 min at 3-7 C and the response was monitored spectrophotometrically. The info presented are the average of 2 split up studies CTEP and shows the amount of uPA activity remaining compared to the neglected SKOV 3 cells. Cell lysates were harvested and protein was separated on the 10 percent polyacrylamide gel, transferred and blotted for active Akt, total Akt, total ERK2 o-r tubulin. Conditioned media from the cells were concentrated using centrifugal selection products having a molecular weight cut-off of 30 kDa and treated just like the lysates but run on either 10 % or 12-pack gels, and blotted for uPA and PAI 1. Densitometry of the Western blots was done with ImageJ computer software from NIH and normalized to either Akt, ERK2, tubulin, whole protein or no therapy, as indicated.