Briefly, DOTAP-chol (20 mM) and plasmid DNA stock solution dilute

Briefly, DOTAP-chol (20 mM) and plasmid DNA stock solution diluted

in 5% dextrose in water (D5W) were mixed in equal volumes to give a final concentration PF-6463922 manufacturer of 4 mM DOTAP-chol, i.e., 150 μg DNA in 300 μL final volume (ratio, 1:2.6). These reagents were diluted and mixed at room temperature. The DNA solution was added to DOTAP-chol liposomes and rapidly mixed by pipetting up and down twice with the pipette tip. The DNA:liposome mixture thus prepared was precipitate-free and used for all the in vivo experiments. The size of the DNA fragments in the DNA:liposome mixture was determined to be in the range of 300-325 nm. Flow cytometric analysis LLC cells were seeded in a 6-well plate and incubated for 24 h, then treated with normal saline (NS), CDDP, Lip-null, Lip-mS, or Lip-mS+CDDP (DNA at 1 μg/mL and CDDP at 4 μg/mL). Forty-eight hours later, the cells were washed with PBS and resuspended in propidium iodide/RNase A solution (0.5 mL), incubated at 37°C for 30 min and analyzed by flow cytometry. Animal studies Studies involving whole mice were approved by the Institute’s Animal Care and Use Committee. Female C57BL/6 mice of 6 to 8 weeks old were purchased from the experimental animal center of Sichuan University (Chengdu, Sichuan Province, China) and challenged subcutaneously (s.c.) with LLC

cells (5 × 105 cells in 50 μL PBS) in the right Selleckchem SNX-5422 flank. Mice were randomly divided into 4 groups (8 mice per group) and treated with NS, Lip-mS, CDDP or Lip-mS + CDDP until the tumors had mean diameter of 3 mm. Lip-mS was injected into mice via the tail vein at 5 μg per day once daily for 10 days (days 0 to 9) and CDDP (made in the Qilu Shandong Medical Factory) was injected into mice via the tail vein at 1 mg/kg per week (days 1, 8). Tumor size was determined by caliper measurement of the largest and perpendicular LEE011 purchase diameters every two days. Tumor volume was calculated according to the formula V = 0.52ab2 (a is the largest superficial diameter and b is the smallest superficial diameter). Protein extraction and

selleckchem Western blot analysis Tumor tissue samples were ground into powder under liquid nitrogen by milling in mortar, and lysed in RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 150 mM NaCl, 1% nonidet P-40 (NP-40), 1 mM EDTA, 1 mM NaF, 1 mM Na3V4, 1 mM phenylmethylsulfonyl fluoride). After being quantifided by Bradford assay, lysates were subjected to 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) electrophoresis, electroblotted with Sartoblot onto a PVDF membrane (Millipore, Bedford, MA) for 1 hr at 100 V, and then membrane blots were blocked at 4°C in 5% non-fat dry milk, washed, and probed with rabbit anti-mouse Caspase 9 antibody (Abcam, Cambridge, United Kingdom) at 1:1000 and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100. The blots were labeled with horseradish peroxidase-conjugated secondary antibody and visualized by chemiluminescence detection.

Comments are closed.