Fig. 4

The Extracellular Domains of Crb2a- or Crb2b-lf-Mediated Cell Aggregation in HEK Cells (A) Unlike wild-type HEK cells, it is more common for monoclonal HEK cells that express full-length Crb2a or Crb2b-lf to grow in large aggregates. (B) Expression of Crb3a, Crb2a-Δ^ex, and Crb2b-Δ^ex, which lack typical Crb extracellular domains, did not prompt HEK cell aggregation. (C) Suppression of N-cadherin by N-cadherin siRNA treatment further reduced the tendency for wild-type HEK cells to form aggregates, suggesting that the low-level cell-cell adhesion in wild-type HEK cells was dependent on N-cadherin. However, loss of N-cadherin did not prevent Crb2a- or Crb2b-lf-expressing HEK cells from aggregating, suggesting that the Crb2-mediated cell-cell adhesion was independent of N-cadherin. (D) Crb2a and Crb2b-lf localized to the cell membrane in HEK cells with various degrees of enrichment at the cell-cell junctions (arrowheads, insets). The cell nuclei were stained with DAPI (blue). (E) To quantitatively evaluate the adhesion property of the extracellular domain of Crb proteins, various Crb-expressing HEK cells were counted and categorized according to their cluster sizes. The total numbers of cells are indicated below the histograms. The symbol Δ^ex indicates that the extracellular domain of the corresponding Crb protein was deleted (Figure 6A). See also Figure S3.