Fig 4. NMII activation and Ca²⁺ elevation are required for GC-induced F-actin reduction at adherent sites of polarized epithelial cells.

(A, B) Non-polarized and polarized T84 cells were inoculated with Pil+Opa+ or ΔOpa GC from the top chamber for 6 h (MOI~10). Cells were fixed and stained for phosphorylated myosin light chain (pMLC) and GC and analyzed using 3D-CFM. Shown are representative xy images of the top surface, xz images crossing both the top and the bottom surface (A), and the FIR (±SEM) of pMLC staining underneath individual GC microcolonies relative to the adjacent no GC surface area (B). (C, D) Non-polarized and polarized T84 cells were incubated from the top chamber with or without GC Pil+Opa+ or Pil+ΔOpa in the presence or absence of thapsigargin (10 μM) for 4 h. Cells were incubated with the Ca²⁺ indicator Fluo4 and the membrane dye CellMask and analyzed using 3D-CFM. Shown are representative xz images (C) and the mean fluorescence intensity (MFI) (±SEM) of Fluo-4 in the cytoplasmic region of individual epithelial cells (D). (E, F) Polarized T84 cells were pre-treated with or without ML-7 (10 μM) or 2APB (10 μM) and apically inoculated with Pil+ΔOpa GC in the presence or absence of inhibitors for 6 h (MOI~10). Cells were fixed and stained for F-actin using phalloidin and GC using antibody and analyzed using 3D-CFM. Shown are representative xy and xz images (E) and the FIR (±SEM) of phalloidin staining underneath individual GC microcolonies relative to the adjacent no GC surface area (F). Data points in (B) and (F) represent individual GC microcolonies. Scale bar, 20 μm. n = 3 three independent experiments. *p<0.05; ***p<0.001.