Sc 783

The slides containing the tumor tissue samples were subjected to antigen retrieval with citrate buffer, and the endogenous peroxidase activity was blocked using 3% hydrogen peroxide in water. For immunohistochemical staining, the slides were incubated with rabbit polyclonal antibodies targeting Bax (sc-6236) and Bcl-2 (sc-783) (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing with PBS, the slides were incubated with biotinylated HRP-conjugated streptavidin secondary antibody (E0432; 1:1,000; Dako, Glostrup, Denmark), prior to being further washed with PBS. The slides were then incubated with DAB, prior to being counterstained with diluted Harris hematoxylin. Following staining, five ×400 magnification fields (DM IL LED Microscope) were randomly selected in each slide, and the average ratio of positive cells in each field was counted using the Image-Pro Plus true color multi-functional cell image analysis management system, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). In order to prevent any non-specific staining, PBS was used as a replacement to the primary antibody in the negative control.

In total, 1×10⁶ MCF-7 cells/well were seeded onto 6-well plates and treated with liriodenine (0, 0.1, 1 and 10 µM) for 48 h. According to the manufacturer's protocol, the cells were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The protein concentrations were determined using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Total protein (50 µg) was isolated with 10% SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skimmed milk powder in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature, and incubated with antibodies against B-cell lymphoma-2 protein (dilution, 1:500; Bcl-2; sc-783), cyclin D1 (dilution, 1:500; sc-717), p53 (dilution, 1:500; sc-6243), vascular endothelial growth factor (dilution, 1:500; VEGF; sc-13083) and β-actin (dilution, 1:500; sc-7210; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The membrane was washed with TBST and incubated with secondary antibody mouse anti-rabbit IgG-HRP (sc-2357, dilution, 1:3,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h, and then assessed by an BeyoECL Plus (P0018; Beyotime Institute of Biotechnology). The optical density was analyzed using Quantity One software (version 3.0; Bio-Rad Laboratories, Inc.).