GSK3 signaling promotes desmosome assembly. (A) Schematic diagram of GSK3 consensus sites (bold) in DP. (B) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6), assessed by immunofluorescence. Bars, 10 µm. (C) NaCl- and LiCl-treated SCC9 cells stained with DP (NW6) and keratin (K8), assessed by confocal immunofluorescence. (C′) Pearson’s correlation coefficients were calculated for DP and keratin. *, P < 0.001. Error bars indicate SEM. (D) LiCl-, Kenpaullone-, and siCtr- or siGSK3-treated SCC9s, assessed by immunofluorescence. (E and E′) NaCl- or LiCl-treated SCC9s transfected with control or caGSK3, assessed by immunofluorescence. DP fluorescence intensity was measured, normalized to background, and plotted. (F) caGSK3 was transfected into SCC9s and assessed by immunoblotting. (G) LiCl or NaCl SCC9s, fixed at 0, 1.0, and 2.5 h after being switched into high calcium media, were assessed by immunofluorescence. (G′) DP fluorescence intensity along cell borders was quantified (>200 cell borders) from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) Monolayers of SCC9 cells stably expressing WT DP-GFP (Godsel et al., 2005) were subjected to scratch wounding, treated with either 40 mM NaCl or LiCl, and imaged. Shown are stills of Videos 1 and 2 at 0 and 80 min. White arrows mark forming borders. (H′) Stills of LiCl treatment in WT DP-GFP cells from Video 3. Green circles denote DP particles appearing and being retained in the cytoplasm in a filamentous alignment. Time points indicate the minutes lapsed from initiation of cell–cell contact. Bars, 10 µm. (H″) Recruitment of DP-GFP to SCC9 cell–cell borders occurs in three temporally overlapping phases (Godsel et al., 2005) in control cells, whereas LiCl treatment decreases DP border intensity. Fluorescent intensities over time were calculated for representative borders from Videos 1 and 2 and correspond to borders shown in H. Results are representative of data obtained from >30 videos for each condition.