N cadherin

Initially, a radio-immunoprecipitation assay (RIPA) lysis buffering solution (Beyotime, Shanghai, China) was applied to abstract proteins. Following the separation of proteins by sodium salt-polyacrylamide gel electrophoresis, target proteins were electrophoretically moved to nitrocellulose films, blocked via 5% milk-TBST for 2 h under room temperature. Subsequently, they were washed three times in TBST buffering solution and cultivated with the first anti-substances, viz., COL5A1, COL1A1, E-cadherin, N-cadherin, snail, GADPH rabbit polyclonal antibody (Proteintech, USA), vimentin, and vinculin mouse polyclonal antibody (Proteintech, USA) at 4°C overnight. Subsequently, the films were washed three times in TBST again, each for 10 min. Goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) (Abcam, USA) were added then for cultivation. Afterwards, the membranes were cleaned in TBST as per the steps mentioned above to analyze relative protein expression with Image-Pro Plus software 6.0.

Total protein in PC cells was extracted using radioimmunoassay precipitation lysis buffer (Beyotime Biotechnology, Suzhou, China) containing phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China). Protein concentrations in the samples were determined using the bicinchoninic acid method (Servicebio, Wuhan, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gels (Meilune, Dalian, China)and thereafter, transferred to polyvinylidene fluoride membranes (Thermo Scientific, USA). The membranes were blocked with skim milk powder (Beyotime Biotechnology, Suzhou, China) and incubated with primary antibodies (GADD45A [1:500], Cat No. A1797, Abconal, China; CDK1 [1:500], Cat No. 19532-1-AP, Proteintech, China; CCNB1 [1:500], Cat No. 28603-1-AP, Proteintech, China; N-cadherin [1:500], Cat No. 22018-1-AP, Proteintech, China; E-cadherin [1:500], Cat No. 20874-1-AP, Proteintech, China; and β-actin [ACTB], 1:500; Cat No. 20536-1-AP, Proteintech) for 16 hours at 4℃. After washing twice with Tris-buffered saline containing 0.1% Tween‐20, the membranes were incubated with secondary antibody and visualized using an enhanced chemiluminescence reagent. ACTB was used as the loading control to calculate the relative protein expression.

Total proteins from cell lines and tissues were extracted with RIPA buffer and then quantified by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulphate–polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase-9 (Proteintech 10380-1-AP), Caspase-3 (Proteintech 66470-2-Ig), Caspase-8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612) and METTL3 (ABclonal A8370).

The total protein of cells at the logarithmic phase was extracted by a lysis buffer. BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to measure the total protein concentration. The harvested samples were heated for 10 min at 100 °C for denaturation. About 30 μg proteins were separated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane (0.45 μm, Millipore, Billerica, MA, USA). The PVDF membrane was blocked with skimmed milk and then incubated with primary antibodies overnight at 4 °C. After incubation with HRP goat anti-rabbit or goat anti-mouse IgG antibody (Proteintech, Wuhan, China), the protein signal was captured with autoradiography films. The E-Cadherin, p-GSK-3β^Ser9 and β-catenin antibodies were all obtained from Cell Signaling Technology. GSK3β, N-Cadherin, c-Myc, CyclinD1 and Gapdh antibodies were purchased from Proteintech, while EphA5 was purchased from Invitrogen. Snail was purchased from R&D Systems.

Western blotting was performed according to previously described standard methods.²⁸ Antibodies were used to against AKR1C2 (Abcam), phospho‐AKT Ser473 (Affinity), total AKT (Proteintech), cleaved‐PARP(Affinity), caspase3 (Cell Signaling Technology), E‐cadherin (Proteintech), N‐cadherin (Proteintech), Vimentin (Proteintech), Androgen receptor (AR) (Invitrogen), GAPDH (Proteintech). Dilution ratio used was according to the manufacturer's instructions. GAPDH was used as an internal reference.

Protein samples from cells were extracted by RIPA buffer (Beyotime, China) and separated in 10% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies (anti-p-AKT-phosphoT308, 1:1000, abcam, England; anti-AKT, 1:1000, abcam, England; anti-p-GSK3β-phosphoS9, 1:1000, abcam, England; anti-GSK3β, 1:1000, abcam, England; cyclinD1, 1:500, Proteintech, China; cyclinE1, 1:500, Proteintech, China; p21, 1:1000, Proteintech, China; β-catenin, 1:500, Proteintech, China; vimentin, 1:500, Proteintech, China; E-cadherin, 1:500, Proteintech, China; N-cadherin, 1:500, Proteintech, China; TAF15, 1:500, Proteintech, China; RAB14, 1:500, abcam, England) overnight and then incubated with the corresponding secondary antibody. Band intensity was measured using chemiluminescence (ECL) system kit according to the manufacturer’s instructions (Solarbio, Beijing, China). The optical densities (OD) value was analyzed with ImageJ software (NIH, Bethesda, MD, USA).