Tight junction structure and formation and basal contact comparison between Jeg-3 wild-type and knockout cells. (A) Apical Z-projections of Jeg-3 wild-type and knockout cells stained with E-cadherin and actin. (B) Still frames from Video 1 of Jeg-3 cells transfected with ZO-1–GFP showing the development of tight junctions. Following cell division, new junctions are formed that look similar between wild-type and knockout cells (yellow arrowheads; 1 h after division). Over time (2.5 h after division), these same junctions (yellow arrowheads) increase in tortuosity in the knockout cells but are maintained as relatively straight (low tortuosity) in the wild-type cells. (C) Western blotting of nonmuscle myosin-II, vinculin, α-actinin, and tubulin in Jeg-3 wild-type and double-knockout cells show similar levels of expression. (D) Immunofluorescence staining of paxillin focal adhesion marker and actin shows similar organization. (E) Immunofluorescence staining of actin and vinculin indicates that localization of vinculin is similar between knockout cells and Jeg-3 wild-type cells. Scale bars, 10 µm in A, 20 µm in B, 10 µm in D, 5 µm in E.