After incubation, the noninvaded cells on the upper membrane surface were removed with a cotton tip, and the cells that passed through the filter were fixed and stained using 0. 1% crystal violet. The sellckchem numbers of invaded cells were counted in five randomly selected high power fields under a microscope. This experiment was performed in triplicate. Matrigel in vitro HUVEC tube formation assay Cells Transfected with control or FoxM1 siRNA were cultured in serum free RPMI 1640 for 24 hours. The conditioned medium were collected, centrifuged and stored at 20 C until assay. HUVEC in 500 ul of the indicated conditioned medium were seeded onto a 24 well plate, which was precoated Inhibitors,Modulators,Libraries with 100 ul of growth factor reduced Matrigel for 30 minutes.
Following stimulation with the cell conditioned medium for 12 hours, Inhibitors,Modulators,Libraries tube formation was observed under an inverted microscope and counted. The number of tube formations was measured by counting the number of tube like structures formed by connected endothelial cells in five Inhibitors,Modulators,Libraries randomly selected fields under a microscope. The assay was performed in triplicate. Statistical analysis The statistical analyses were performed using the Statis tical Package for the Social Sciences, version 16. 0. A paired samples t test was used to compare FoxM1 mRNA and protein expression in the ccRCC tissues with that of their paired adjacent nontumor tissue samples. The relationship between FoxM1 protein expression and the clinicopathological features was analyzed using ��2 tests. Overall survival curves were calculated with the Kaplan Meier method and were analyzed with the log rank test.
A Cox propor tionalhazards analysis was used in univariate and multi variate analyses to explore the effects of FoxM1 expression and ccRCC clinicopathological variables on survival. Unpaired 2 tailed Students t tests were used to analyze comparisons between the 2 groups. A P value of 0. 05 was regarded as statistically significant. Results FoxM1 Inhibitors,Modulators,Libraries mRNA and protein expression in primary ccRCC tissue samples and RCC cell lines We first examined FoxM1 mRNA expression in 39 paired clinical samples from ccRCC patients by real time quantitative PCR. The results revealed a statistically significant elevation of FoxM1 mRNA in tumors, as compared to the matched adjacent nontumor tissues. To investigate whether FoxM1 was also elevated at the protein level, western blot was performed on those 39 paired ccRCC clinical samples.
We found that the protein level of FoxM1 in tumor tis sues was significantly higher than that in adjacent non tumor tissues, consistent Inhibitors,Modulators,Libraries with the results of real time quantitative PCR. The protein level of FoxM1 in four representative pairs of samples is shown in Figure 1C. We also used real time quantitative PCR and western blot to detect the expression of FoxM1 mRNA and protein in RCC cell lines as well as in an immortalized Sorafenib normal human proximal tubule epithelial cell line.