Fig. 2

RhoA upregulation is essential for apical junction formation. a Assay of RhoA activity using the lysates of HT29 cells treated or not treated with nocodazole. Active and total RhoA were detected by immunoblotting for anti-RhoA antibody. The graph shows the ratio of active to total RhoA, assessed by densitometric scanning of blots. n = 5 independent experiments, mean ± SEM. b Immunostaining for RhoA and E-cadherin in HT29 cells transfected with control (CTRL) or RhoA siRNA (si). c Lateral views of HT29 cells immunostained as in b. d Staining for ZO-1, E-cadherin and DNA in HT29 cells transfected with CTRL si or RhoA si. Relative ZO-1 positive areas are also shown. n = 65, 65, 71, 70, 66 and 71 fields for nocodazole-untreated (control) and -treated cells, which were transfected with CTRL si, RhoA si #1 and RhoA si #2, respectively, pooled from two independent experiments. **P = 0.0011, ****P < 0.0001 by two-tailed Mann–Whitney U-test. e Immunostaining for GEF-H1 and α-tubulin in HT29 cells transfected with GEF-H1 siRNA. Images were obtained with Airyscan. Part of the control image is enlarged at the right. f Staining for ZO-1, E-cadherin and DNA in HT29 cells transfected with CTRL or GEF-H1 siRNAs. Relative ZO-1 positive areas are also shown. n = 69, 67, 68, 68, 69, and 72 fields for nocodazole-untreated and -treated cells, which were transfected with CTRL si, GEF-H1 si #1 or GEF-H1 si #2, respectively, pooled from two independent experiments. ****P < 0.0001 by two-tailed Mann–Whitney U-test. NS, not significant. Scale bars, 10 μm. See also Supplementary Fig. 2