Primer sequences for IGFBP7 (fw: 5′-GTAAGGAGGACGCTGGAGAGT-3′,
rev: 5′-CTGGCTGTAATAAAGTGTTAGTGG-3′) and β-actin (fw: 5′-CCGTGAAAAGTGACCCAG-3′ rev: 5′-TAGCCACGCTCGGTCAGG-3′). PCR and gelelectrophoresis conditions were described as previous . The expected size of fragment of IGFBP7 and β-actin was 255 bp, 136 bp, respectively. Analysis of Cell Viability Cell viability was determined by the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, MK5108 cell line Japan) and measured by microplate reader scanning at 450 nm as previously described elsewhere . Quantification of cell apoptosis by flow cytometry B16-F10 cells were washed by PBS and collected after digestion by 0.25% trypsin, cell suspension was added dropwise selleck kinase inhibitor to PBS while gently vortexed, then centrifuged at 1000 rpm at 4°C for 10 min. After resuspension of the cells in labeling buffer, 10 μl Annexin VFITC was added and then incubated in the dark. Following 150 μL of propidium iodide (PI) was added, the cells were incubated for 2 h at room temperature. Then cell apoptosis was measured by flow cytometry [16, 17]. Mice
Thirty-six six-week-old female Wild-type C57BL/6J mice weighing 18-25 g were treated in accordance with the guidelines of the National Institutes of Health for the humane treatment of animals, and all animal protocols were approved by Huazhong University of Science and Technology’s animal care and use committee. Mice were anesthetized with urethane (1.9 g/kg sc; 12.5 mg urethane/ml 0.9% saline; PAK6 Sigma Chemical, St. Louis, MO), and their temperature was maintained at 37°C. 1 × 104 B16-F10 cells were injected subcutaneously in the lower backs of mice, where MM emerged after 1 week. Tumor volume (v) was calculated as follow, v = L × I2 × 0.52, where L and I represent the selleck maximum and minimum tumor diameter measured
weekly. All the mice were divided into three groups randomly (n = 12 each group), termed pcDNA3.1-IGFBP7, pcDNA3.1-CONTROL and B16-F10 cells groups respectively. Then Invivofectamine reagent-plasmid duplex complexes 200 μl (Reagent for in vivo plasmid delivery, Invitrogen, U.S.A), containing pcDNA3.1-IGFBP7 (1 μg), or pcDNA3.1-CONTROL (1 μg), DMEM 200 μl were respectively injected into the tumors for every 3 day. The delivery efficiency was evaluated by GFP fluorescence and RT-PCR. After 3 weeks the mice were killed (with permission of the Animal Protection Association of Tongji Medical College). Tumors were cryosectioned or fixed in 10% buffered formalin and embedded in paraffin detected by immunohistochemistry. Western blot analysis IGFBP7 expression changes within mouse xenografts were checked by western blotting as described previously , the antibodies to IGFBP7 and β-actin were purchased from (R&D systems U.S.A.).