Figure 1. DUR is required for strong cell–cell adhesion. (A) Schematic representation of Dsg2 and Dsg2 mutants. P, precursor sequence; EC, extracellular cadherin repeat; EA, extracellular anchoring domain; TM, transmembrane domain; IA, intracellular anchoring domain; ICS, intracellular cadherin-like sequence; L, linker domain; RUD, repeat unit domain; TD, terminal domain. (B, a) SCC68 or SCC9 cells expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or a nonspecific sequence (control [Ctl]). 48 h after transfection, cell lysates were collected and immunoblotted with the indicated antibodies. The epitope for 4B2 is in the C terminus of Dsg2 tail and therefore recognizes endogenous Dsg2 and Dsg2.FL-GFP but not Dsg2.ICS-GFP. (b) SCC68 cells stably expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or a nonspecific sequence (control KD). 24 h after transfection, cells were placed in medium containing 1.0 mM Ca2+. Another 24 h later, at which time the cells were confluent, a dispase assay was performed. Bar, 5 mm. The graph shows the number of fragments counted from one representative experiment with three replicates. This experiment was repeated twice. Error bars represent mean ± SEM. (c) SCC68 or SCC9 cells expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or nonspecific sequence (control KD). 24 h after transfection, cells were placed in medium containing 1.0 mM Ca2+. Another 24 h later, cells were stained for DP (red) and imaged using an N-SIM system. Bar, 1 µm. (C) Mutant expressing SCC68 cells grown in medium with 0.09 mM Ca2+ were lysed in 0.5% Triton X-100 buffer. Supernatants (S) were collected after centrifugation. To the remaining pellets (P), USB equal to the volume of the supernatant was added, and pellets were solubilized. Equal amount of supernatant and pellet samples from each mutant were blotted for FLAG. Similar results were obtained for cells in 1.0 mM Ca2+.
Figure 1.

DUR is required for strong cell–cell adhesion. (A) Schematic representation of Dsg2 and Dsg2 mutants. P, precursor sequence; EC, extracellular cadherin repeat; EA, extracellular anchoring domain; TM, transmembrane domain; IA, intracellular anchoring domain; ICS, intracellular cadherin-like sequence; L, linker domain; RUD, repeat unit domain; TD, terminal domain. (B, a) SCC68 or SCC9 cells expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or a nonspecific sequence (control [Ctl]). 48 h after transfection, cell lysates were collected and immunoblotted with the indicated antibodies. The epitope for 4B2 is in the C terminus of Dsg2 tail and therefore recognizes endogenous Dsg2 and Dsg2.FL-GFP but not Dsg2.ICS-GFP. (b) SCC68 cells stably expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or a nonspecific sequence (control KD). 24 h after transfection, cells were placed in medium containing 1.0 mM Ca2+. Another 24 h later, at which time the cells were confluent, a dispase assay was performed. Bar, 5 mm. The graph shows the number of fragments counted from one representative experiment with three replicates. This experiment was repeated twice. Error bars represent mean ± SEM. (c) SCC68 or SCC9 cells expressing Dsg2.ICS-GFP or Dsg2.FL-GFP were transfected with 20 nM siRNA oligos targeting endogenous Dsg2 (Dsg2) or nonspecific sequence (control KD). 24 h after transfection, cells were placed in medium containing 1.0 mM Ca2+. Another 24 h later, cells were stained for DP (red) and imaged using an N-SIM system. Bar, 1 µm. (C) Mutant expressing SCC68 cells grown in medium with 0.09 mM Ca2+ were lysed in 0.5% Triton X-100 buffer. Supernatants (S) were collected after centrifugation. To the remaining pellets (P), USB equal to the volume of the supernatant was added, and pellets were solubilized. Equal amount of supernatant and pellet samples from each mutant were blotted for FLAG. Similar results were obtained for cells in 1.0 mM Ca2+.

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