E cadherin

Total protein was harvested and extracted from NSCLC tissue samples and cell lines (SPC-A1 and A549) with RIPA buffer (Millipore, Bedford, MA, U.S.A.) and the protein concentration was calculated with reagent kit via Bradford method (Beyotime, Shanghai, China). Then, protein lysates were isolated via 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and then transfected onto polyvinylidene fluoride (0.45 µm, PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin (BSA; Absin Bioscience, Shanghai, China) in 1× Tris-buffered Saline Tween-20 (TBST; Absin Bioscience), and then incubated with specific diluted primary antibodies, including E-cadherin (1:1000, ab76055), Vimentin (1:1000, ab8979), N-cadherin (1:500, ab18203), and MTDH (1:800, ab45338), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, ab8245) as endogenous control, and all primary antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). After the membranes were incubated overnight at 4°C, 1× TBST was used to wash membranes for thrice, followed by covering the corresponding second antibody. Finally, protein bands were visualized using ECL Reagent (Millipore).

Stable OVCA429 cell lines were inoculated on sterilized coverslips in a 12-well cell culture plate. After fixation with 4% paraformaldehyde (5 min), the cells were washed three times with PBS buffer (5 min each time) and permeabilized with 0.5% TritonX-100 for 10 min. 1% BSA was added to block the antigen for 30 min. The primary antibody FSP1 (abs138156, Absin, China; 1:200) and E-cadherin (abs158305, Absin, China; 1:200) were incubated with the samples overnight at 4 °C in a humidified chamber. After incubation, the coverslips were washed with PBS and incubated with Alexa Fluor 594 (abs20021, Absin, China; 1:100) and Alexa Fluor 488 (abs20014, Absin; 1:100) fluorescently labeled secondary antibodies at room temperature in a dark box for 1 h. The nuclei were stained with DAPI before covering the coverslips with anti-fluorescence quenching mounting medium (P0126, Beyotime, China). The slides were observed and recorded using a confocal fluorescence microscope (Olympus, FV3000, Japan).