Figure 3.

Cyclin B1 protein is upregulated by UCHL1. (A) Expression of cyclin proteins four days after siRNA transfection of ARK1, ARK2 and HEC-50 cells. Representative experiment (3 biological replicates). (B) qRT-PCR quantification of cyclin B1 RNA expression four days after siRNA-mediated UCHL1 silencing. Statistical significance was determined by the T-Test (two-tailed, equal variance). Error bars represent mean ± SD (3 biological replicates). * p < 0.05. (C) Cyclin B1 protein expressions in ARK2 and HEC-50 cells stably overexpressing UCHL1. Representative experiment (3 biological replicates). (D) qRT-PCR quantification of cyclin B1 RNA expression in ARK2 and HEC-50 cells stably overexpressing UCHL1. Error bars represent mean ± SD (3 biological replicates). (E) Cyclin B1 protein levels across histological subtypes of endometrial cancer (TCGA data set). Statistical significance was determined by the Kruskal-Wallis test. Error bars represent median with interquartile range. ** p < 0.01, *** p < 0.001. (F) Correlation between cyclin B1 positivity and UCHL1 staining intensity in the USC cohort. Statistical significance was determined by the Spearman’s test. Error bars represent mean ± SD. (G) Representative slides of samples stained for both cyclin B1 and UCHL1 with increasing semiquantitative score. Scale bar, 100 µm. (H) Immunohistochemical staining of cyclin B1 and UCHL1 in tumors established by intraperitoneal injection of nude mice with ARK1 cells transduced with doxycycline-inducible control shRNA (n = 7) or anti-UCHL1 shRNA (n = 5). Representative slides. Scale bar, 100 µm. (I) Percentage positivity of cyclin B1 in tumor cells at time of sacrifice. Statistical significance was determined by the Mann-Whitney test. Error bars represent mean ± SD. * p < 0.05. The uncropped blots and molecular weight markers are shown in Supplementary Materials.