Validation of the vinculin-dependent junctional reinforcement of mitotic cell–cell junctions. (A) Immunostaining of MDCK cells expressing WT- or ΔVBS α-catenin for endogenous paxillin, β-catenin, and ZO-1, showing that focal adhesions (FA) are maintained in α-catenin^ΔVBS cells, and no apparent differences in adherens junctions (AJ) and tight junctions (TJ) in α-catenin^ΔVBS cells compared with WT cells during interphase. (B) WB of lysates from parental-, vinculin knockdown (KD)-, and vinculin KD with addback of mCherry-vinculin MDCK cells probed for vinculin and α-tubulin. (C) Immunostainings of MDCK cells with vinculin knockdown (KD), and vinculin KD with addback of mCherry-vinculin, for α-catenin and α-tubulin to visualize mitotic cells and the integrity of cell–cell contacts. Blue arrowhead indicates a disrupted cell–cell contact. (D) Quantification of the fraction of mitotic and interphase cells in which cell–cell contacts are perturbed, in MDCK monolayers with vinculin KD, and vinculin KD with addback of mCherry-vinculin. Data were pooled from three independent experiments, with at least 25 cells analyzed per condition in each experiment. Gray bars represent the mean and SD of the average fraction of disrupted cell–cell contacts in the independent experiments. **, P = 0.0021; ***, P = 0.0008; n.s., not significant (P = 0.12, P = 0.10 respectively); paired t test. (E) Representative x,z-projection of a polarized MDCK monolayer cultured under conditions used for the permeability assay (Fig. 4, C and D), in which apicobasal polarization is indicated by the apical actin brush border that is visualized with SiR-Actin. Scale bars, 10 µm. IB, immunoblot; mC, mCherry; Vin, vinculin.