Bovine serum albumin (bsa)

For immunofluorescence, cells cultured on coverslips were briefly washed by PBS once, followed by fixation in cold methanol at − 20 °C for 10 min or in 4% paraformaldehyde (PFA) in PBS at 37 °C for 15 min. After washed with PBS, cells were permeabilized with PBS containing 0.5% Triton X-100 and 2% bovine serum albumin (BSA, Genview, Florid, USA) or 10% horse serum for 1 h. Cells were then incubated with the primary antibody diluted in PBS containing 1% BSA or 1% horse serum at 4 °C overnight followed by incubation with Alexa Fluor coupled secondary antibody diluted in PBS containing 1% BSA or 1% horse serum at room temperature for 90 min. Cells were stained with DAPI to visualize the nucleus before slides were mounted with Fluoromount-G anti-fade mounting medium (SounthernBiotech, 0100–35, Alabama, USA).
All the samples were observed at room temperature under a fluorescence microscope (ECLIPSE 80i; Nikon, Tokyo, Japan) equipped with a 40 × 0.75 NA objective lens (Nikon) or a confocal microscope (TCS SP8; Leica, Wetzlar, Germany) equipped with 63 × 1.4 NA and 100 × 1.4 NA objective lens (Leica). For imaging of the colocalization of CCP5-GFP with γ-tubulin or CP110, the HyD detector and Lightning process were used. Images were acquired using NIS-Elements software (Nikon) or Las X software (Leica). Image processing was performed using ImageJ and Photoshop (Adobe, California, USA).

Protein extraction and quantitation were performed using kits obtained from KeyGEN BioTECH, China. Supernatants were resolved on 10% SDS/PAGE and transferred to 0.45 μm PVDF membranes (Millipore, Billerica, MA). Membranes were blocked with 5% Bovine Serum Albumin (GENVIEW, USA) for 1 h at room temperature and probed with the indicated primary antibodies at 4°C overnight. Membranes were then washed and labeled with secondary antibodies for 2 h at room temperature. Blots were visualized using the BIO-RAD imaging system (BIO-RAD, Hercules, CA, USA). The following antibodies were used: FEN1 (A1175, ABclonal, China), E-cadherin (ab40772, Abcam, Cambridge, UK), N-cadherin (ab76057, Abcam, Cambridge, UK), Vimentin (ab92547, Abcam, Cambridge, UK), MMP2 (ab92536, Abcam, Cambridge, UK), MMP9 (ab76003, Abcam, Cambridge, UK), Anti-rabbit IgG, HRP-linked Antibodies (#7074, Cell Signaling Technology, Danvers, MA, USA). GAPDH was used as a loading control (#5174, Cell Signaling Technology, Danvers, MA, USA).

The films were plated into 24-well plates and cultured with 5 × 104 cells per well in an incubator for 24 h. Subsequently, the cells were treated with plasmids and drugs for 48 h. Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton-X-100 at room temperature for 15 min. After blocking with 5% bovine serum albumin (GENView, United States) for 1 h, the cells were incubated with primary antibodies against α-SMA (1:100, Santa Cruz) 50 μl at 4°C overnight. Subsequently, cell nuclei were stained with DAPI for 1 min. We observed cells using fluorescence microscopy (Olympus AX70, Japan). The entire experimental process was carried out in the dark.