Next, the cells were washed once with PBS PI was added to sample

Next, the cells were washed once with PBS. PI was added to samples at a final concentration of 15 mol l, and after 5 min of incubation, the cells were analyzed Wortmannin PI3K inhibitor using flow cytometry. The percentages of the nuclei in CNE 2 cells at each phase of the cell cycle were calculated using the MultiCycle software program. Immunoblot Analysis Protein analysis using immunoblotting and immunopre cipitation was performed with primary antibodies against p53, p21, c Myc, cyclin E, cyclin D1, and actin as described previously. Total cell lysates were har vested, electrophoresed using 12% sodium dodecyl sul fate polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes.

Immunoblotting was per formed using the primary antibodies described above followed by detection of protein expression using second ary antibodies conjugated with horseradish peroxidase, and Inhibitors,Modulators,Libraries blots were developed using ECL chemiluminescent reagent. RNA Interference Transient small interfering RNA transfection was performed using Lipofectamine 2000 and 50 nM siRNA oligonucleotides. Commer cially purchased siRNAs were scrambled, glyc eraldehyde 3 phosphate dehydrogenase siRNA, and c Myc siRNA. The siRNA duplexes were introduced into CNE 2 cells according to the siRNA manufacturers protocol. After transfection with siRNA for 48 h, cells were harvested for immunoblots and cell cycle analysis. The scrambled siRNA construct was used as a negative control. In vivo treatment and immunohistochemistry assay Four week old athymic nude mice obtained from the Animal Inhibitors,Modulators,Libraries Center of Southern Medical University received subcutaneous injection of 1 107 CNE 2 cells in each axillary area.

When subcutane ous tumors developed to more than 1,500 mg, mice were euthanized and tumors were dissected and mechanically dissociated into equal pieces Inhibitors,Modulators,Libraries to be transplanted into the flank areas of a new group of mice. When xenograft tumors became palpable, mice were ran domly divided into control and ApoG2 groups. Each group contained 8 mice, and there was no difference in tumor size between groups. Based on our labs policy, when xenograft tumors developed to more than 1,000 mg, mice were euthanized and tumors were dissected and weighed. Immunohistochemical analysis was performed on tissue sample sections of CNE 2 xenografts obtained from control and ApoG2.

All samples were stained with hematoxylin and eosin and microscopically examined to confirm the CNE 2 cell origin. Sections were then stained with c Myc at 4 C overnight and then vis ualized using diaminobenzidine as peroxidase substrates. Statistical analysis All analyses to compare the Inhibitors,Modulators,Libraries significance of measured lev els were completed using the unpaired t test by SPSS 16. 0 software. Inhibitors,Modulators,Libraries Results ApoG2 unlike Inhibits Cell Proliferation of NPC cells Our previous work demonstrated that ApoG2 could significantly kill NPC cells and suppress the growth of NPC xenografts in nude mice.

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