Prmt5

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%).

Cells were plated on coverslips for 24 h prior to transfection with siRNAs or DNA plasmids. After experimental treatment, cells were fixed for 30 min in 4% PFA, washed with PBS three times, and permeabilized and blocked with 0.25% Triton X-100 and 10% BSA in PBS for 1 h. Cells were then incubated with primary antibody overnight at 4 °C and with fluorescence-conjugated secondary antibody for 1 h at room temperature. After DAPI (Thermo Fisher Scientific, Waltham, MA) staining for 5 min, cells were mounted onto glass slides. Staining was assessed using a Zeiss LSM 710 immunofluorescence microscope and ZEN software (Carl Zeiss, Oberkochen, Germany). Cells with ≥5 foci per cell were classified as foci-positive. A minimum of 300 cells were counted for each experimental repeat. Representative images were obtained at ×100 magnification. The fluorescence intensity of pictures was analyzed by Image Studio version 5.0. Antibodies used include γH2AX (Cell Signaling Technology, #2577, 1:2000), 53BP1 (Bethyl Laboratories, A300-272, 1:2000), and PRMT5 (Millipore, 07–405, 1:1000).

Furamidine, pentamidine, hexamidine, and TC-E5003 were purchased from Tocris. Decamidine and SKLB639 were described previously^{15 (link),17 (link)}. Antibodies used in this study include PRMT1^{3 (link)}, PRMT5 (Millipore), aDMA (Cell Signaling), sDMA (Millipore), MYCN^{3 (link)}, TH (Millipore), PHOX2B (Abcam), PARP (Cell Signaling), ATF5 (Abcam), Flag (Thermo Fisher Scientific), and β-actin^{3 (link)}.