Do 7 m7001

The expression of p53 was immunohistochemically examined in all patients who were positive for S-p53Ab preoperatively and in representative patients (20 patients) who were negative for S-p53Ab preoperatively. This test was performed as described previously [8 (link), 9 (link)]. Briefly, immunohistochemical staining was performed using mouse anti-human p53 monoclonal antibody (DO-7 M7001, Dako, Glostrup, Denmark) on 4 μm thick serial sections of formalin-fixed, paraffin-embedded colorectal cancer. A tumor in which more than 10% of the 1200~1800 counted cell nuclei were stained was considered to be positive (at ×400). The results were evaluated by two independent researchers (Figure 1).

The Ishikawa cells were obtained from Dr. Katsutoshi Oda (The University of Tokyo) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco), and maintained at 37 °C in 5% CO₂. Liraglutide was purchased from Novo Nordisk (Bagsværd, Denmark), and AICAR was purchased from WAKO chemicals (Osaka, Japan). Mouse monoclonal antibody anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-LC3 (M152–3, MBL, Nagoya, Japan), anti-p62 (M162–3, MBL), anti-p53 (DO-7, M7001, DAKO, Glostrup, Denmark), anti-estrogen receptor (ER; 1D5, M7047, DAKO), and anti-progesterone (PR; PgR636, M3569, DAKO) were purchased and used for this study. Rabbit polyclonal antibody against GLP-1R (bs-1559R) was purchased from Bioss ANTIBODIES (Boston, MA, USA). Rabbit monoclonal antibodies against AMPK (5831) and p-AMPK (4188) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and sheep anti-mouse IgG secondary antibodies for Western blotting were purchased from GE Healthcare (Little Chalfont, Buckingamshire, UK), and the FITC-conjugated donkey anti-mouse IgG antibody for immunofluorescence was purchased from Santa Cruz.