ROCK inhibitors rescue epithelial morphology in HCT8 cells. (A) Confocal images of HCT8-INAVA-GFP cells treated with ROCK inhibitors Y27632 (10 μM), Y39983 (10 μM), or myosin II inhibitor blebbistatin (50 μM) for 1 h. Scale bar = 10 μm. (B) Quantification of INAVA-GFP localized to lateral membranes, n = 120–150 cells/condition. (C) Confocal images of HCT8 WT and HCT8-INAVA-GFP cells treated as in A and stained with tight junction marker ZO-1. Scale bar = 10 μm. (D) Brightfield images of HCT8 WT and HCT8-INAVA-GFP cysts grown in Matrigel ± Y27632 (10 μM). Overview scale bar = 500 μm. Enlarged image scale bar = 100 μm. (E) Relative distribution of HCT8 cyst morphology from D, n = 80–98 cysts/condition. (F) Dose response of ROCK inhibitor Y39983, Y27632, or blebbistatin (left to right, 33 μM, 6.7 μM, 1.3 μM, 0.3 μM) in blocking IL-1β–induced condensates, mean ± SEM, n = 2. (G) HCT8-INAVA-GFP cells with doxycycline-inducible GEF mutant myc-ARNO-(E156K). Cells were treated overnight with doxycycline (1 μg/ml), fixed, stained, and imaged at four positions/well at 40× magnification, n = 2. Scale bar = 10 μm. (H) Confocal images (left) and quantification (right) of puncta inhibition as in G but treated with IL-1β (10 ng/ml) for 60 min. Scale bar = 20 μm, mean ± SEM, n = 2. *, P < 0.05; **, P < 0.01.