Anti n cadherin

For EMT markers detection, HCEs were fixed with 3.5% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA, sigma, USA), and incubated over night at 4 °C with primary antibodies as following: anti-N-cadherin (1:100, Abclonal, China), anti-E-cadherin (1:100) and anti-vimentin (1:100). After washing with phosphate buffered saline (PBS, Gibco), the cells were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated mouse immunoglobulin G secondary antibody (1:200). The stained cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA) and viewed under a consistent fluorescence in situ hybridization (FISH) imager (BX51, Olympus, Tokyo, Japan).

Total protein samples were retrieved from bladder tissues or cultured cells, and their quantity was assessed by a BCA Protein Assay Kit (Invitrogen) after processing in RIPA lysis buffer supplied with protease and phosphatase inhibitors [20 (link), 21 (link)]. For western blot analysis, a 10% SDS-PAGE gel and 30 μg of proteins were packed and transported to a PVDF membrane. Next, the membranes were blocked for 1 h at room temperature with 5% milk before incubation with primary antibodies: anti-GREM1 (#PK12868; 1:1000; Abmart), anti-E-Cadherin (#A20798; 1:1000; Abclonal), anti-N-cadherin (#A19083; 1:1000; Abclonal), anti-Vimentin (#A19607; 1:1000; Abclonal), anti-p-PI3K (#17366; 1:1000; CST), anti-PI3K (#T40064; 1:1000; Abmart), anti-p-AKT (#4060; 1:1000; CST), anti-AKT (#60203; 1:5000; Proteintech) or anti-GAPDH (#AC001; 1:5000; Abclonal) at 4 °C overnight. The following day, the membranes were incubated with peroxidase-conjugated secondary antibody at room temperature for 1 h, pictured by the chemiluminescence system (ChemiDocTM Touch; Bio-Rad, USA), and assessed with the ImageJ program.

RIPA buffer (Servicebio, China) was used to extract total protein from tissues or cells, and the protein concentration was determined using a bicinchoninic acid assay kit (Boster, China). Protein samples were subjected to electrophoresis and transferred onto a polyvinylidene fluoride membrane. The protein blots were then incubated with the specified primary antibody overnight at 4 °C, as following: anti-α-SMA, anti-Collagen I, anti-N-cadherin, anti-Fibronectin, anti-E-cadherin, anti-Vimentin, anti-β-actin, anti-LC3, anti-p62 and anti-GAPDH (all from Abclonal, China), anti-TFEB, anti-ATP6V0C, anti-CDKN1A, anti-CCNB1, anti-STX17, anti-SNAP29, anti-VAMP8, anti-DNMT3a and anti-DNMT3b (all from Proteintech, USA), anti-p-CDK1 and anti-DNMT1 (both from CST, USA) antibodies, and subsequently incubated with the appropriate secondary antibody horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) (SAB, USA) for 1h. Blots were visualized through the enhanced chemiluminescence detection system (Bio-Rad, USA).