N cad

For immunohistochemical staining, 4-μm-thick sections received antigen retrieval and endogenous peroxidase activity blocking. Then, the samples were incubated in goat serum for 30 min at room temperature to block the nonspecific antigen, followed by incubating in primary antibodies against K10 (1:200; Santa Cruz), K14 (1:200; Santa Cruz), and p-AKT (1:250; Affinity Biosciences) overnight at 4 ℃. Finally, the immune reactivities of the sections were determined using the HRP-streptavidin detection system (ZSGB-bio, Beijing, China). Images from above-mentioned sections were acquired with a BX63 fluorescence microscope (Olympus).
For immunofluorescence staining, 4-μm-thick sections received antigen retrieval and permeabilizing with 0.5% Triton X-100. After blocking with goat serum (Solarbio), primary antibodies against Ki67 (1:250; Affinity Biosciences), Cytokeratin (Pan) (1:200; Huabio, Hangzhou, China), E-cad (1:200; Affinity Biosciences), Vim (1:500; Abcam), and N-cad (1:200; Santa Cruz) were loaded and kept overnight at 4 ℃. Then, Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Abcam) were loaded at room temperature for 2 h. The nuclei were stained with DAPI (Beyotime), with the images visualized via the LSM-980 confocal microscope (Carl Zeiss).

Whole-cell protein was extracted with the RIPA lysis buffer containing protease inhibitor (Beyotime) and protein phosphatase inhibitor (Beyotime) on ice. Equal amounts of protein (30 μg) were loaded into 10% (w/v) SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, St. Louis, MO, USA), with the membrane then incubated with primary antibodies at 4 °C overnight, followed by three series of TBST washing and 2 h of HRP-conjugated secondary antibodies incubation at room temperature. The proteins were visualized through chemiluminescence and imaged on a Gelview 6000 Pro (BLT Co., Ltd, Guangzhou, China). The protein bands were quantified by densitometry analysis using Image J software.
The primary antibodies used in this study included antibodies against GAPDH (1:1000; Affinity Biosciences), Vim (1:1000; Abcam), E-cad (1:1000; Affinity Biosciences), N-cad (1:1000; Santa Cruz, Cincinnati, OH, USA), PI3K (1:1000; Santa Cruz), AKT (1:1000; Santa Cruz), p-PI3K (1:1000; Affinity Biosciences), p-AKT (1:1000; Affinity Biosciences), and PTHR1 (1:1000; Affinity Biosciences).