Cdkn2b

The human gastric adenocarcinoma cancer cell lines MGC803, BGC823, MKN45, AGS and SGC7901 and the normal gastric epithelium cell line (GES-1) were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). Western blot analysis was conducted according to our previous protocol.^{24 (link)} Antibodies used in the study were: E2F1 (cat. # ab14768, Abcam, Hong Kong, China), CDKN2B (cat. # sc-271791, Santa Cruz, Dallas, TX, USA), STAU1 (03-116, Millipore, Bedford, MA, USA), FLAG-tagged antibodies (8146 S, Cell Signaling Technology, Boston, MA, USA), and GAPDH antibody was used as control.

This study was approved by the Ethics Committee of the The Affiliated Cancer Hospital of Nanjing Medical University, while informed consents were provided by all the patients. Surgically derived CRC biopsies were fixed in formalin solutions (10%; v/v), embedded in paraffin and sectioned at 3 ~ 5 μm thickness. The slides were washed with 3% hydrogen peroxide (H₂O₂; v/v) to block endogenous peroxidase activity and to avoid immunoreaction. Subsequently, the slides were incubated with the primary antibodies more than 4 hours, including PRMT5 (Sigma, P0493), EZH2 (ProteinTech, 21800-1-AP) and CDKN2B (Abcam, ab53034). After washing with PBS, the sections were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (HRP; Sigma-Aldrich) and 3, 3'-diaminobenzidine (DAB; Pierce) Substrate solution to visualize the immunoreaction. The staining of CRC slides were reviewed by two trained pathologists blindly and independently. Semi-quantitative of protein expression was calculated according to staining intensity and percentage of positive cells to generate a histological score (H score; range: 0 ~ 300). H score = ΣPi (i + 1), where is the intensity of staining (0 ~ 3) and Pi is the percentage of staining positive cells (0% ~ 100%).