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For in vitro experiments conditioned medium was collected after a culture period of 48 hours. For long stimulation experiments CM was collected after a culture period of 4 days and subsequently diluted 1:3 with serum reduced medium. Cell viability and proliferation assay HUVEC and G55 Ivacaftor 873054-44-5 cells were seeded on 48 well tissue culture plates and incubated in basal medium or in CM or mixtures of CM from PAE WT, PAE Tum and PAE ES cells additionally containing 4% FCS. Each stimulation experiment was performed in triplicate. After 24 and 48 hours of incubation at 37 C, cells were trypsinized and counted using the Vi Cell XR. Cellular viability and proliferation was assessed using the WST 1 assay following the manufacturer´s instructions. Stably PRLR transfected or mock transfected G55 WT or G55 cells were cultured under serum deprivation in presence of AG490 and or 2 nM prolactin. To quantify cell viability, cells were incubated with WST 1 Panobinostat LBH589 reagent for 1 h and the absorbance was measured using a plate reader at 450 nm. Each stimulation experiment was performed in quintuplicate. Cell viability of experimental cells was related to cell viability of control cells, which was set to 100%. Apoptosis assay G55 cells were seeded at subconfluent density into multi well tissue culture plates. After culture overnight, cells were washed twice with PBS, and medium was replaced with CM from PAE WT, PAE Tum or PAE ES cells, or a mixture of CM from PAE Tum and PAE ES cells. Incuba tions of cultures were continued for 24 hours before cells were analysed for apoptosis. For analysis, adherent cells were detached LY2109761 価格 and pooled with floating cells. Apoptosis was assessed by flow cytometric analysis of cells stained with FITC conjugated annexin V and PI. Values represent the mean of three independent experiments. Western blotting Supernatants of transfected PAE cells were tested for transgene expression using Western blot analysis. CM from PAE ES cells was incubated overnight at 4 C with heparin agarose for pro tein concentration. Supernatant of PAE Tum cells were concentrated overnight at 4 C by Nickel Cam HC resin according to manufacturer instructions. ES, Tum and PRLR were detected by murine ES poly clonal antibody, His probe polyclonal antibody and HA antibody, re spectively. The signal was visualized by Lumigen PS 3 detection reagent and exposed to an Amersham Hyperfilm ECL. In vitro wound healing assay HDMEC cells were cultured in 24 well tissue culture plates in endothelial growth medium with sup plements. After reaching confluence each well was scratched with a standardized pipette tip, resulting in an EC free wound. Medium was replaced with CM of WT or transfected PAE cells additionally supplemented with 4% FCS. Photographs of each well were taken direct after scratching and after 20 hours incubation. The width of the gap was determined using AxioVision40 V4. 8 software and values representing the closing wound were compared between experimental groups. Values represent the mean of three independent experiments. In vivo tumor model Animal experiments were conducted according to the UKCCR guidelines for the welfare of animals in experi mental neoplasia.