Cyt c

Protein samples (30 μg) were adjusted to an identical volume and 4 μl 6X loading buffer was added prior to samples being heated for denaturation (95°C). The proteins were separated on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis gels, transferred onto a polyvinylidene difluoride membrane and the membrane was blocked with blocking buffer (membranes and buffer from Solarbio Science & Technology Co., Ltd.) at 4°C for 1 h. The membrane was incubated with primary antibodies against Cyt C (1:3,000; Bioss, Shanghai, China) rat monoclonal PARP (1:3,000; sc-71851, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C and then a horseradish peroxidase enzyme-labeled secondary antibody (1:1,000) at 37℃ for 1 h. The protein expression levels were determined using an Odyssey CLX system (LI-COR Biosciences, Lincoln, NE, USA).

The Day 28 was considered as an experimental endpoint. The tumors were weighed after mice were sacrificed. The removed tumors were used to prepare paraffin sections, which were then performed immunofluorescent (IF) staining for terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick end labeling (TUNEL) (Roche, Germany) and immunohistochemical (IHC) staining of tumor apoptosis-related markers: p53 (1:400), Cyt C (1:400), Bcl-2 (1:400), which were obtained from Bioss (China) and Bax (1:100), which was purchased from Proteintech (USA). Quantification data were performed by calculating the mean optical density value of positive-staining areas versus the whole areas in five randomly selected fields using Image-Pro Plus software (Media Cybernetics).