Zo 1 antibody

We used coverslips to grow cells in six-well culture plates, with cells cultured for 24 h. We then treated the cells with FSK (50 µM) or 0.05% dimethyl sulfoxide (DMSO) for 24, 48, or 72 h. Cells on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by blocking and permeabilization with the Animal Free Blocker (Vector Laboratories, Burlingame, CA, USA) containing 0.05% saponin for 20 min at room temperature. Cells were then stained with mouse monoclonal anti-zonula occludens protein-1 (ZO-1) antibody (Cat No. ab216880, Abcam, 1:100) and Alexa Fluor 488-conjugated anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, USA). Stained specimens were mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories) and examined with a LSM700 laser scanning confocal microscope and the LSM software ZEN 2012 (Carl Zeiss, Jena, Germany). For each group we counted the numbers of fused cells in 8–10 microscopic fields that were randomly selected. In each field, we counted both the total number of nuclei and the numbers of nuclei in STB-like fused cells. We then calculated the percentages of fused cells as the ratio of the number of nuclei in the fused cells to the total number of nuclei in the microscopic field.

Cells were fixed in 4% paraformaldehyde, sealed with Immnol Fluorence Staining Secondary Antibody Dilution Buffer (Beyotime), and then incubated with a 1:200 dilution of ZO-1 antibody (ab96587, Abcam) at 4°C for 24 h. After washing, cells were incubated in a 1:200 dilution of FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime) for 30 min at 37°C. DAPI was prepared for nuclei staining at a 1:1000 dilution for 5 min. Images were captured with confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Cell fluorescence was analyzed by Image J software (Rawak Software, Inc. Germany).