For validation, real time RT PCR was performed using customer reviews a SensiMix SYBR No ROX Kit and a Rotor Gene 6000 detection system. Real time cell proliferation and migration assay Real time cell proliferation and migration experiments were performed using the RTCA DP instrument, which was placed in a humidified in cubator maintained at a 5% CO2 at 37 C. For proliferation assay cells were seeded in complete medium in 16 well plates at density of 5,000 cellswell. The plate containing gold microelectrodes on its bottom was monitored every 10 minutes for 4 hours, then once every 30 min, until the end of experiment, which was in total 72 hours. Cell migration was performed using special 16 well plates with 8 um pores. These plates, resembling conventional transwells, have microelectrodes placed on the underside of the membrane.
Cells were seeded into the upper chamber at a density of 20,000 cellswell in a serum free medium and the lower chamber was filled with complete medium. The plate was monitored every 15 minutes for 12 hours. Data analysis was performed using RTCA software Inhibitors,Modulators,Libraries 1. 2 supplied with the instrument. Senescence associated beta galactosidase activity assay Cells were fixed for 5 min at room temperature and rinsed several times in PBS. To measure SA B gal activity, cells were in cubated in a staining solution for 24 h at 37 C. Cells were washed and embedded in PBS, viewed in an inverted transmission microscope and photographed. Chicken chorioallantoic membrane xenograft model On embryo development day 0 fertilized chicken eggs were placed in a 75 Inhibitors,Modulators,Libraries 80% humidified 37 C incubator to allow normal embryo development.
On day 3 eggs were opened, egg shells removed and embryos were placed Inhibitors,Modulators,Libraries in a sterile Petri dish in Inhibitors,Modulators,Libraries an egg incubator to induce CAM development. On day 8, when chorioallantoic membrane and its vasculature were well developed, all experiments were performed. HMECs were transfected one day before the ex periment either by EpCAM adenoviruses or GFP control adenoviruses. 3. 0 105 cells were resus pended in a 30 uL drop of ice cold growth factor reduced Matrigel containing TGF B1 in a con centration of 1. 7 ngmL and the mixture solidified for 30 min at 37 C. Subsequently, 4 onplants per chicken were grafted on the CAM. Growth of HMECs onplants was inspected on a daily basis using a stereo fluorescence microscope.
On day 6 post grafting chicken embryos were sacrificed with hypothermia, xeno grafts cut out and stored either in 4% paraformaldehyde for immunohistochemical studies Inhibitors,Modulators,Libraries or in TRI reagent for RNA isolation. Statistical analyses Statistical analyses were performed with the GraphPad Prism 5. 0 software for Windows. All tests of statistical significance were two sided. Students T test, two definitely way ANOVA and Mann Whitney U Tests were used to study differences between two groups. Statistical analyses of quantitative PCR data were performed according to the delta Ct method de scribed by Pfaffl et al.