Ab76011

Total proteins of each cultured cell were extracted with RIPA Lysis Buffer (Applygen Technologies Inc., Beijing, China). After the protein concentration was determined by BCA kit (MultiSciences, Hangzhou, China), the target proteins were classified by 15% or 8% SDS-PAGE (MultiSciences) and then transferred to PVDF membranes (Millipore, Darmstadt, Germany) and blocked with 5% skimmed milk powder at 25°C for 90 minutes. The membranes were incubated overnight at 4°C with primary antibodies, anti-E-Cadherin (ab40772, 1 : 20000), anti-N-Cadherin (ab76011, 1 : 10000), anti-HMGA1 (ab168260, 1 : 1000), and anti-GAPDH (ab8245, 1 : 5000) (Abcam, Cambridge, UK). The membranes were then incubated with HRP-conjugated secondary antibodies (1 : 1000, MultiSciences) at 25°C for 90 min, and the bands were visualized by a chemiluminescence.

Total proteins in HTR-8/SVneo cells after the indicated treatment were isolated with RIPA buffer. The protein concentration was detected by using a bicinchoninic acid (BCA) protein assay kit (Invitrogen, USA). After being subjected to a 12% gel sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then impeded with 5% skim milk for 2 h followed by incubation with primary antibodies against Bcl-2 (ab32124; 1:1000; Abcam, China), Bax (ab32503; 1:1000; Abcam), cleaved-caspase3 (ab32042; 1:500; Abcam), MMP2 (ab92536; 1:1000; Abcam), MMP9 (ab76003; 1:1000; Abcam), E-cadherin (ab40772; 1:10000; Abcam), N-cadherin (ab76011; 1:5000; Abcam), p-p38 (mAb #4511; 1:000; Cell Signaling Technology, USA), p-ERK1/2 (mAb #4370; 1:2000; Cell Signaling Technology), p-JNK (mAb #4668; 1:1000; Cell Signaling Technology), p38 (mAb #8690; 1:1000; Cell Signaling Technology), ERK1/2 (mAb #8544; 1:1000; Cell Signaling Technology), JNK (ab76125; 1:1000; Abcam) and GAPDH (ab8245; 1:500; Abcam) at 4˚C overnight. Thereafter, the membranes were washed with tris-buffered saline and Tween (TBST) for three times, followed by incubation of membranes with HRP-labeled secondary antibody. Finally, the protein bands were captured by improved chemiluminescence (ECL, USA).

Polyclonal antibodies anti-E-cadherin (#ab40772, abcam), anti-N-cadherin (#ab76011, abcam), actin reference antibody (#ab5694, abcam), and second antibody (goat anti-rabbit lgG, #ab205718) were purchased from Abcam. Western blotting was carried out following standard procedures described previously 18 (link).

The cells were lysed using the Radioimmunoprecipitation Assay lysis buffer containing 1% phenylmethanesulfonyl fluoride prior to centrifugation at 14,000 g and 4°C for 30 min to extract the total protein content, followed by quantification using the Bradford Method Protein Assay Kit (Beyotime, Shanghai, China). Next, the protein content was boiled for 5 min, cooled on ice, and centrifuged for 30 s. The supernatant liquid was isolated using sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes at 100 V. After a membrane blockade with 5% skim milk at room temperature, the membranes were cultured with the corresponding primary antibodies at 4°C for 12 h. Subsequently, the membranes were rinsed with tris buffered saline tween twice for incubation with luciferase-labeled goat anti-rabbit IgG (at a dilution ratio of 1:2500, ab6721, Abcam) at room temperature for 1 h. After 3 rinses, the membranes were visualized using the enhanced chemiluminescence and photographed with a membrane scanner. The included primary antibodies were as follows: N-cadherin (at a dilution ratio of 1:1000, ab76011, Abcam), E-cadherin (at a dilution ratio of 1:1000, ab40772, Abcam), and GAPDH (at a dilution ratio of 1:1000, ab9485, Abcam).

Cells were lysed with radioimmunoprecipitation analysis buffer (Sigma-Aldrich) containing protease inhibitor. Total proteins were determined by bicinchoninic acid (BCA) method (Sigma-Aldrich). Proteins were separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Sigma-Aldrich). After being blocked with 5% skim milk at room temperature for 1 h, the membrane was incubated with primary antibodies overnight. Primary antibodies were rabbit anti-TBX3 (1 : 1000, ab154828, Abcam, UK), rabbit anti-E-cadherin (1 : 10000, ab40772, Abcam, UK), rabbit anti-N-cadherin (1 : 5000, ab76011, Abcam, UK), rabbit anti-Vimentin (1 : 1000, ab92547, Abcam, UK), and rabbit anti-β-actin (1 : 1000, ab8227, Abcam, UK). After being washed with PBS with Tween 20 3 times (each time for 10 min), the membrane was reacted with secondary antibody goat anti-rabbit IgG H&L coupled with horseradish peroxidase (HRP) (ab205718, Abcam, UK) for 1 h at room temperature. Enhanced chemiluminescence (Solarbio, Beijing, China) was added for development. Analysis was taken using the gel imager (Gel Doc XR, Bio-rad, USA), and grey scale value was processed by ImageJ.

Total protein from cells was extracted with RIPA buffer (abs9229, Absin, China). The extracted protein was centrifuged and then quantified with the BCA kit (pc0020, Solarbio, China). After electrophoresis, they were electrotransferred onto the nitrocellulose membrane. Then, 5% nonfat milk was taken to block the membrane. After rinsing, the blocked membrane was subjected to primary antibodies at 4°C all night. The next day, an antirabbit secondary antibody (ab7090, Abcam, UK) was added. After reacting at 37°C for 1 h, a color reagent (1705061, BIO-RAD, USA) was taken to visualize the blots. Finally, the blots were developed in ChemiScope 3300 mini equipment (Clinx, China). The primary antibodies of collagen II (1: 1000, ab34712), fibronectin (1: 1000, ab268020), α-SMA (1: 50000, ab124964), Notch1 (1: 2000, ab52627), Jag1 (1: 500, ab7771), HEY1 (1: 3000, ab154077), HES1 (1: 1000, ab108937), TGF-β (1: 1000, ab215715), E-cadherin (1: 50000, ab40772), vimentin (1: 5000, ab92547), N-cadherin (1: 20000, ab76011), collagen I (1: 1000, ab260043), and GAPDH (ab181602) were bought from Abcam (UK).

Briefly, the samples were dehydrated with xylene and alcohol. Antigen retrieval was performed in 10 mmol/L sodium citrate solution (pH 6.0) at 100°C for 16 min, and the samples were cooled for 30 min. Endogenous peroxidase activity using 3% hydrogen peroxide and blocked with foetal bovine serum for 30 min. Thereafter, the slides were incubated with anti-ADCY6 rabbit polyclonal antibodies (ab14781, 1:500, Abcam, USA), anti-N-cadherin (ab76011, 1:100, Abcam, USA), anti-vimentin (ab92547, 1:200, Abcam, USA), anti-Ki-67(ab16667, 1:200,Abcam, USA), anti-E-cadherin(ab40772, 1:500, Abcam, USA) and anti-twist1(ab175430,1:200, Abcam, USA) at 4°C overnight. Then, the samples incubated with biotinylated secondary antibody at 37°C for 30 min. The samples were stained with DAB (3, 3- diaminobenzidine) and Mayer’s haematoxylin.
ADCY6 staining classifications were as followings: range 0–3: 0, negative; 1, weak; 2, moderate; and 3, strong, the percentage of positive cells range 0–4: 0, negative or <5%; 1, 6%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%. Used the percentage of positive cells and the intensity to determine the final staining scores. Grades <4 were defined as low ADCY6 expression, while Grades ≥4 were defined as high ADCY6 expression.