Fig. 4.

Altered cell cycle and signaling pathways. (A) Urothelial cells from the transgenic mice (6-week old; n = 3) were subject to fluorescein-activated cell sorting (upper panel) or western blotting using anti-CDK4 or anti-CDK6 antibodies (lower panel). Note the marked increase of S phase diploid and aneuploid cells in Ras*/SV40T double transgenic mice. (B) Ras-GTPase assay. In vitro assay of Ras-GTPase was carried out using total urothelial proteins extracted from the transgenic strains (all 6 weeks of age). Values were means ± SD. Note that the Ras activity was considerably higher in Ras* and Ras*/SV40T mice (n = 8 per genotype). (C and D) Western blotting and immunohistochemical detection (anti-p-S6) of signaling pathway components in urothelial cells from the transgenic strains (all 6 weeks of age). Note that the MAPK pathway activation was primarily associated with Ras* activation, whereas AKT-mTOR pathway activation was highly activated in SV40T mice and even more so in Ras*/SV40T mice. All panels in D are ×200.