N1icd

Total protein was extracted from treated cells with whole cell lysis assay (KeyGen Biotech Co., Ltd, Jiangsu, China). The concentration of protein was determined with BCA protein quantitation assay (KeyGen Biotech Co., Ltd, Jiangsu, China). The 5× SDS-PAGE loading buffer (Solarbio Science & Technology Co., Ltd, Beijing, China.) was added to the protein solution and denatured in metal bath at 100°C for 8 min. The treated protein was separated by 12% SDS-PAGE and transferred to 0.2 μM PVDF membrane and closed by PBST of 5% skimmed milk for 2 h at room temperature. Then, the primary antibodies were incubated overnight at 4°C, and after washing the membrane with PBST, the secondary rabbit anti-mouse antibodies (Cell Signaling Technology, Inc, Danvers, Massachusetts, USA.) were incubated for 2 h at room temperature and finally protein expression assays were performed on ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Inc, Shanghai, China) with ECL detection kit (KeyGen Biotech Co., Ltd, Jiangsu, China.). β-Actin was used as an endogenous reference. The primary antibodies were Ab against HES1, HE5, Notch1, N1ICD (Abcam Plc, Shanghai, China.), and actin (Cell Signaling Technology, Inc, Danvers, Massachusetts, USA.).

Formalin-fixed, paraffin-embedded tissue samples were cut into 4-μm-thick sections. Antigen retrieval was performed using a pressure cooker for 3 min in 0.01 M citrate buffer (pH 6.0). DU145 and 22RV1 cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature followed by treatment with 0.05% Triton X-100 at 4 °C for 5 min. Sections and cells were blocked with 1% BSA for 1.5 h at room temperature and incubated with primary antibodies against CD68 (1:500; Trevigen), CD206 (1:500; Abcam, London, UK), N1ICD (1:1000; Abcam, London, UK), E-cadherin (1:500; Novus, Centennial, USA) or N-cadherin (1:500; Novus, Centennial, USA) at 4 °C overnight. The samples were incubated with secondary antibodies conjugated with Alexa Fluor or HRP for 1 h at room temperature. Tissues or cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to detect nuclei and visualized by fluorescence microscopy. For IHC, the secondary antibody was diluted to 1:750 for recognizing primary antibodies. The staining for IHC was visualized using the VECTASTAIN ABC peroxidase system and peroxidase substrate DAB kit.

Cells were lysed with RIPA buffer (Millipore, Temecula, CA, USA) containing protease and phosphatase inhibitor cocktail (1:100) with 2 mM PMSF. The protein concentration was determined by using bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). 30 μg of total protein was used for this assay. Protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then it was transferred to 0.45 μm Nitrocellulose Membrane (Millipore, CA, USA).
After the overnight incubation with primary antibodies at 4°C, the membrane is hybridized with secondary antibody (1:4,000 dilution, Epitomics, China) at room temperature for 1 h. Protein signals were detected by ECL system. The primary antibodies were listed as follows: Sirt3 (1:1,000 dilution, Cell Signaling Technology, Danvers, USA); Notch1 (1:1,000 dilution, Cell Signaling Technology, Danvers, USA); Foxo3a (1:1,000, Cell Signaling Technology, Danvers, USA); β-Tubulin (1:1,000 dilution, Abcam, Hong Kong); N1ICD (1:1,000 dilution, Abcam, Hong Kong).