Cells were seeded at low density (400 cells in six-well plates)

and allowed 10 days to form colonies, which were stained and manually counted. The results are presented in Figure 3B. Ku-0059436 chemical structure Consistent with the proliferation assays, PACE4 and PC7 knockdown cells formed significantly fewer colonies than the NT control cells (42% and 40%, respectively), and no significant changes were observed for the furin and PC5/6 knockdown cells. As the cell culture environment has the obvious limitations of in vitro experiments, the physiological context was then considered in an effort to validate the obtained cell proliferation and clonogenicity results. Each knockdown cell line was subcutaneously xenografted on athymic nude mice, and tumor volumes were monitored over time. Mean tumor volumes were determined and plotted ( Figure 4, A and B). As previously reported, a tumor latency phase was observed before

the tumors reached an exponential growth phase [17]. Interestingly, in contrast with the results from the in vitro assays, only the PACE4 knockdown cell–derived xenografts had a statistically significant lower growth rate when compared to control NT cells (37% overall reduction of tumor sizes). Moreover, the PC7 knockdown xenograft behavior was strikingly http://www.selleckchem.com/GSK-3.html different when compared to the in vitro assay as their tumor growth rates were significantly higher than the growth rates of the control tumors (29% overall increase in tumor sizes). Consistent with the in vitro assays, the growth rates of both furin and PC5/6 knockdown tumors remained unchanged. At the end of the experiment, the mice were killed, and tumors were excised and weighed. The average tumor weights are reported in Figure 4C. Consistent with their growth rates, PC7 knockdown–derived tumors had significantly higher

weights (250 ± 30 mg) than the PACE4 knockdown–derived tumors, which were significantly lower (100 ± 20 mg) when compared to the control tumors (170 ± 20 mg). No significant changes in tumor weights were observed for the furin and PC5/6 knockdowns (averages of 170 and 150 mg, respectively). Molecular markers were analyzed by IHC in xenografts to evaluate the biologic processes of proliferation that might clarify the growth disparity between in vitro and in vivo www.selleck.co.jp/products/MG132.html conditions. Analyses were performed on excised xenograft sections with the Ki67 proliferation marker, which stains nuclei and allows the proliferating cells to be discriminated. Thus, the determination of Ki67-positive nuclei provided insights supporting tumor growth behavior. The results presented in Figure 5A indicated that cell proliferation indexes among the PC knockdown cell–derived xenografts were equal compared to the NT controls with the exception of PACE4 knockdown cell–derived xenografts, which had a significantly lower index (70%), and furin knockdown cell–derived xenografts, where only a slight but statistically significant difference was observed (87%).