Knockdown of p27 disrupts VHL-mediated tight junction formation. (A) RCC10 cells stably expressing WT pVHL30 were infected with retroviruses containing shRNA constructs directed at luciferase as control (sh Luc) or p27 (sh p27). Approximately 2 weeks post-infection, cell lysates were equally loaded and subjected to p27 and α-tubulin western blotting. (B) RCC10 WT VHL cells infected with shRNA to luciferase or p27 were grown to confluence on collagen I and photographed by digital phase microscopy (original magnification of 100×). (C) RCC10 WT VHL cells infected with shRNA to luciferase or p27 were grown to confluence on coverslips and immunostaining for ZO-1 (left panels) was performed (original magnification of 1000×). DAPI labeled nuclei of corresponding cells are also shown (right panels). (D and E) 786-O cells stably expressing pVHL19 were infected and analyzed as in (A) and (C).