SGEF KD stimulates actomyosin contractility. (A) Confocal images showing maximum projection of apical Z-sections in CTRL, SGEF KD, and Rescue WT MDCK cells stained for endogenous ZO-1 (green), myosin IIB (red), and mNeon-SGEF (magenta, in Rescue). mNeon signal is shown in magenta in Rescue panel to maintain color consistency. Scale bar, 5 µm. (B) Confocal images showing CTRL, SGEF KD, and Rescue WT MDCK cells stained for endogenous F-actin using phalloidin (green) and myosin IIB (red). Left panel: Maximum projection of apical Z-planes; right panel: maximum projection of basal Z-planes. Images were processed using the HyVolution deconvolution package (see Materials and methods). Scale bars, 0.5 µm. (C) Quantification of intensities of ZO-1 at junctions measured using a rectangle of 2 × 3 µm placed along BCJs. At least two fields from two independent experiments were used for quantification (≥100 junctions). Error bars represent SEM. ****, P < 0.00005; ns, nonsignificant using the Mann–Whitney U test. (D) Total cell lysates from confluent CTRL, SGEF KD, and Rescue WT MDCK cells were immunoblotted with ZO-1, myosin IIB, and afadin antibodies. Tubulin was used as a loading control. (E) Maximum projection of confocal images showing the localization of endogenous afadin in CTRL, SGEF KD, and Rescue WT cells. Scale bar, 5 µm. (F) Quantification of the ratio of TCJ over BCJ intensity of afadin was measured as described in Materials and methods. At least three fields from two independent experiments were used for quantification (≥200 junctions). Error bars represent SEM. ****, P < 0.00005; ns, nonsignificant using the Mann–Whitney U test.