Prism version 4

Unpaired t tests and graphs were produced using Prism version 4 (GraphPad). Z-tests were completed in excel. P < 0.05 was considered significant.

Kaplan-Meier curves were assembled in Prism version 4 (GraphPad software). Mean survival times were estimated from Kaplan-Meier curves. Comparisons between survival curves were made by using the logrank test. Correlations between immunohistochemical scores were calculated using Pearson's correlation co-efficient and significance assessed using with Student's t-test. All calculations were performed using SPSS version 21 (IBM).

Data from four independent experiments are reported as the mean ± SEM. Statistical analyses were performed using unpaired two‐tailed Student’s t‐tests with prism, version 4 (GraphPad Software Inc., San Diego, CA, USA). P < 0.05 was considered statistically significant.

The variables were tested for normality by Shapiro–Wilk test. For normally distributed variables, a repeated-measures t-test was used and data were expressed as mean ± standard deviation. When the variables were not normally distributed, Wilcoxon signed-rank test was applied and data were presented as median (min-max). A 2-tailed p-value <0.05 was considered statistically significant. (Graphpad prism, version 4). Spearman’s correlation coefficient is a statistical measure of the strength of a monotonic relationship between paired data.

The results were expressed as means ± standard deviation (SD). Statistical significance of difference between groups was analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using statistical software (Prism,version 4; GraphPad Software Inc., San Diego, CA, USA).

The inhibitory activities of the compounds on human or mouse 11β-HSD1 and 11β-HSD2 were determined using the scintillation proximity assay (SPA). The full-length cDNAs of human or murine11β-HSD1 and 11β-HSD2 were isolated from the cDNA libraries provided by NIH Mammalian Gene Collection. The cDNAs were cloned into pcDNA3 expression vectors. HEK-293 cells were transfected with the pcDNA3-derived expression plasmid and selected by cultivation in the presence of 700 μg/ml of G418. The microsomal fraction overexpressing 11β-HSD1 or 11β-HSD2 was prepared from the HEK-293 cells, which were stably transfected with 11β-HSD1 or 11β-HSD2. The fraction was then used as the enzyme source for SPA. Microsomes containing human or mouse 11β-HSD1 were incubated with NADPH and [³H] cortisone. The product, [³H] cortisol, was specifically captured by a monoclonal antibody coupled to protein A-coated SPA beads. The 11β-HSD2 screening was performed by incubating 11β-HSD2 microsomes with [³H] cortisol and NAD + and monitoring substrate disappearance. All tests were done twice with glycyrrhizinic acid as a positive control. IC₅₀ (X + SD, n = 2) values were calculated by using Prism Version 4 (GraphPad Software, SanDiego, CA).