Knockdown of KHC impairs Dsg2 accumulation at the plasma membrane in Scc9 cells. (A) Scc9 cells in media with 1.2 mM Ca²⁺ were transfected with siRNA oligos against KHC or control nontargeted (NT) siRNA (siRNA_NT) for 2 d, replated and, after 24 h, stained with MitoTracker (Mito), fixed, and colabeled either for Dsg2, Dsc2, or E-cadherin (E-cad). Knockdown of kinesin-1 (indicated by mitochondria collapse, marked by asterisks) inhibits Dsg2 recruitment into the newly forming cell–cell interfaces but does not inhibit Dsc2 and E-cadherin accumulation. Level of knockdown in this experiment was comparable with that in Fig. 2 F. Bars, 20 µm. (B) Line scan analyses of border intensities for Dsg2, Dsc2, and E-cadherin were performed for ∼50 borders per each condition with three scans per border (n = ∼150 for each condition). Borders were chosen randomly from three independent experiments per condition. KHC knockdown results in loss of peak intensity for Dsg2 at cell–cell borders (P < 0.001) and decreased total pixel intensity of Dsc2 staining of ∼10% (P < 0.01). (C) Scc9 cells were transfected with siRNA oligos against KHC or nontargeting siRNA and were cell surface biotinylated 3 d after transfection. Biotinylated proteins were isolated by streptavidin pull-down, and levels of cell surface biotinylated Dsg2, Dsc2, and E-cadherin were identified by immunoblotting (compiled data from the same blot). Average of four independent experiments demonstrates a significant decrease in Dsg2 (*, P < 0.05) but not Dsc2 or E-cadherin cell surface expression. Black lines indicate that intervening lanes have been spliced out. Error bars represent SEM. pxl, pixel.