Fig 3. GC induce differential activation and redistribution of ezrin in non-polarized and polarized epithelial cells for actin reorganization and GC invasion.
Non-polarized and polarized T84 were pretreated with or without the ezrin activation inhibitor NSC668394 (20 μM) for 1 h and apically inoculated with Pil+Opa+, OpaCEA, or ΔOpa GC (MOI~10) for 6 h with or without the inhibitor. (A, B, F, G) Cells were fixed, permeabilized, stained for ezrin (A), F-actin (F), and GC, and analyzed using 3D-CFM. Shown are representative xy and xz images (A, F). The levels of ezrin (B) and F-actin (G) redistribution were quantified by FIR (±SEM) underneath GC microcolonies relatively to the adjacent no GC surface area. Data points represent individual GC microcolonies. (C-E) Cells were lysed and analyzed by western blot. Shown are representative blots (C). The average fold of increase in the ezrin pT567 to ezrin ratio (D) and the ezrin to β-tubulin ratio (E) (±SEM) was quantified by NIH ImageJ. Data points represent individual transwells. (H) Invaded GC (±SEM) were quantified by gentamicin resistance assay 6 h post-inoculation with or without NSC668394 treatment. Data points represent individual transwells. Scale bar, 20 μm. n = 2~3 two to three independent experiments. *p<0.05; **p< 0.01; ***p<0.001.