Cultured Sun2^−/− keratinocytes form altered, mechanically weak intercellular adhesions. (A and B) WT or Sun2^−/− MKCs in high Ca²⁺ medium after 72 h revealed desmosome (DPI/II) and AJ (E-cad) formation. (B) Insets from A show adhesion formation in WT and Sun2^−/− MKCs (arrowheads). (C) Western blot analysis of Triton X-100–soluble and –insoluble fractions of WT and Sun2^−/− MKC cell lysates, cultured in low or high Ca²⁺ medium, and probed for desmoglein 3 (Dsg3), desmoplakin I/II (DPI/II), and β-actin (loading control). (D) DPI/II (red), E-cad (green), and nuclear (Hoechst) localization in WT and Sun2^−/− MKCs after 24 h in high Ca²⁺ medium revealed a uniform distribution of DPI/II along interior and lateral junctions in WT cells. This pattern of localization was perturbed in Sun2^−/− cells. Insets display single lateral adhesions indicated by white boxes. (E) Diagram illustrating the location of interior (magenta) and lateral (blue) cell–cell adhesions in MKCs at the edge of colonies. Black arrow indicates direction of measurements in H. (F) The ratio of interior junction DPI/II intensity per micrometer to lateral junction DPI/II intensity per micrometer in WT and Sun2^−/− MKCs after 24 h in high Ca²⁺ medium or for WT cells pretreated with DMSO or nocodazole before Ca²⁺ addition. Both Sun2^−/− and nocodazole-treated MKCs displayed a significantly increased distribution of DPI/II at interior junctions. n > 67 cells per genotype and condition from three experiments. Statistical significance determined by unpaired, two-tailed t test. The bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the mean. (G) Averaged line scans of DPI/II intensity (arbitrary units [AU]) measured orthogonally across WT and Sun2^−/− interior junctions for images as in D. n > 26 cells per genotype, three measurements per adhesion. (H) Line scans of DPI/II intensity (arbitrary units) along single representative WT and Sun2^−/− MKC lateral adhesions, extending from the interior junctions to the cell edge (representative of n > 106 junctions from three experiments). (I and J) WT monolayers remained intact (I), whereas Sun2^−/− monolayers fragmented drastically (J) after 72 h in high Ca²⁺ medium followed by mechanical challenge. (K) Quantification of the adhesion integrity assay in I and J. Error bars indicate SD for two replicates of three monolayers per genotype. Statistical significance determined by unpaired, two-tailed t test. AU, arbitrary unit; Noco, nocodazole.