Fig. 3

Bex1 localizes to nucleoli and apical side of cells in the cell density-dependent manner. a Immunostaining of BEX at different cell densities in ARPE19 cells. At a low cell density, BEX1 localized to nucleoli, which were stained with a fibrillarin (FBL) antibody. At a high cell density, BEX1 showed a condensed pattern that colocalized with α-tubulin. The inset shows the localization of the BEX1 condensate (arrowhead) to tubulin fibres. Nuclei were stained with DAPI. Scale bar = 25 μm. b Reconstructed z-axis view of ARPE19 cells at the high cell density. In control knockdown cells (siCtrl), the immunostaining showed localization of BEX1 to the apical side of cells where tubulin was enriched (arrowhead). In BEX1-depleted cells (siBEX1), the enrichment of tubulin at the apical surface was lost and cells were flattened. c Reconstructed z-axis view of ARPE19 cells showing the stratification provoked by BEX1 knockdown. Arrowheads indicate the cells overriding other cells. Scale bar = 25 μm. d Cell scattering was induced by BEX1 knockdown. Immunostaining of cell-cell junction protein α-catenin and β-catenin and cytoskeletal protein Vimentin and Filamin. ARPE19 cells were transfected with control siRNA or BEX1 siRNA 96 h prior to the staining. Nuclei were counterstained with DAPI. Scale bar = 25 μm. e Immunostaining of Bex1 at different cell densities in MDCK cells. At a low cell density, Bex1 showed localization to nucleoli which were stained with FBL antibody. At a high cell density, Bex1 showed a condensed pattern which colocalized to α-tubulin. Nuclei were stained with DAPI. Scale bar = 25 μm. f The XY view and the corresponding reconstructed z-axis view of MDCK cells at the high cell density. The immunostaining showed localization of BEX1 to the apical side of cells where tubulin was enriched. Scale bar = 25 μm