*2.11. Immunofluorescent Staining*

Cells with a inoculum density of 1 × 10<sup>5</sup> cells/well were cultured onto coverslips (24-well plate, Fisher Scientific) pre-coated with collagen and fixed with 4% PFA after treatments as described above. The cells were washed with PBS 2 times and incubated with 0.3% Triton X-100 in PBS (PBS/Triton) at room temperature for 10 min. For β-actin staining, the treated cells were incubated with Phalloidin, CF488A (1:100 dilution in PBS, Biotium, Inc., Fremont, CA, USA) at room temperature for 1 h. The cells were then washed three times with PBS and mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). For ZO-1 staining, cells were blocked with 5% goa<sup>t</sup> serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h and then incubated with an anti-rabbit ZO-1 polyclonal antibody (cat. # 61-7300, 1:100 dilution, Thermal Scientific, Ottawa, Canada) at 4 ◦C overnight. The cells were then washed three times with PBS and incubated with an Alexa fluor 488 goa<sup>t</sup> anti-rabbit antibody (Thermal Scientific, cat. # A-11034) for 1 h at room temperature. Rinsed cells were mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories, Inc.)**.** The images were taken by a Zeiss Fluorescence Microscope (Carl Zeiss Ltd., Toronto, ON, Canada) [10].

