Fig. 6
Inactivation of Rac1 reverses EMT in CGN c.3560C > T HT-29 cancer cells and inhibits the growth of CGN c.3560C > T mutant orthotopic colorectal tumors. A Western blot analyses of GTP-Rac1, EMT markers, and β-catenin in CGN WT and c.3560C > T HT-29 cells with or without NSC23766 (100 μM) and EGF (100 ng/ml) treatment. B Densitometric quantification of the expression level of EMT markers assessed by Western blot. Column, mean ± SEM from analysis of three different clones. C (Left) Transwell assay of CGN WT and c.3560C > T HT-29 cells with or without NSC23766 (100 μM) treatment. (Right) Quantitative analyses of the invasion activity of CGN WT and c.3560C > T HT-29 cells with or without NSC23766 (100 μM) treatment. Column, mean ± SEM from analysis of three different clones. Scale bar: 200 μm. D (Left) Migratory activities of CGN WT or c.3560C > T HT-29 cells with or without NSC23766 treatment assessed by using gap closure assay. (Right) Quantitative analyses of the migration activity of CGN WT and c.3560C > T HT-29 cells with or without NSC23766 (100 μM) treatment were assessed by using a gap closure assay. Gap closure area quantified by using the ImageJ software was taken as the index of cell migration activity. Scale bar: 200 μm. Column, mean ± SEM from analysis of three different clones. E Representative images showing tumor growth in CGN WT and c.3560C > T HT-29 orthotopic xenograft tumor receiving saline or NSC23766 treatment were assessed by IVIS system. Mice were repeatedly imaged until week four after inoculation to record luminescence signals. The data were shown as radiance (photons/ sec/ cm2/ steradian) with a color bar. F Quantitative analysis of luminescence intensity from each tumor. Mice were analyzed by optical bioluminescence imaging at 1, 7, 14, 21, and 28 days after cancer cell injection. Arrows indicate the treatment time points. Value, mean ± SEM, n = 6 in each group. G Representative image of immunohistochemistry staining for GTP-Rac1 from xenograft tumor tissues. H The expression level of GTP-Rac1 was detected and analyzed by HistoQuest software in tumor tissues. Column, mean ± SEM from analysis of three different clones. t-test for statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001