SH3BP1 depletion disrupts epithelial cell–cell junctions. (A and B) Caco-2 cells were transfected with control and Cdc42-directed siRNAs (50 nM) and then analyzed by immunoblotting (A) and immunofluorescence (B). Shown are epifluorescence images. (C) Caco-2 cells were transfected with myc-tagged Cdc42L61, a constitutively active mutant, and then stained for ZO-1 and the transfected protein. (D) Summary of RhoGTPases and GAPs identified in the functional siRNA screen (see also Table S1). (E–L) Caco-2 cells were transfected with siRNAs as indicated and then analyzed by immunofluorescence (E and G–L) or immunoblotting (F). Apart from F-actin, shown are images of markers for tight junctions (E and G), adherens junctions (H), and basolateral and apical cell surface domains (K). Shown are epifluorescence (D and F–K) and confocal (L, xy and z sections are provided) images. For F-actin, apical and basal focal planes are shown. Panel I shows a quantification of the effect on the subcellular distribution of F-actin (counting cells with strongly induced basal F-actin; mean basal actin intensity increased by >60% in SH3BP1-depleted cells) and three junctional markers (counting cells with discontinuous, irregular junctional staining). Shown are means ± 1 SD, representing the cells in at least five different fields per condition (n ≥ 5). Note that assembly of tight and adherens junctions is affected, but different components are affected to different extents. (M–O) Complementation of SH3BP1 siRNA transfection with a siRNA-resistant cDNA. Caco-2 cells were first transfected with siRNAs as indicated and then, after 3 d, with a plasmid carrying the siRNA-resistant cDNA. Cells were then analyzed by immunoblotting (M) or immunofluorescence (N). Panel O shows a quantification of the ZO-1 staining (six fields for each condition). The arrowheads in N point to staining for myc-SH3BP1 along cell–cell contacts. Error bars show SDs. Bars, 10 µm. p, phosphorylated.