After these genes were screened out we continued to measure their expression levels in the xenografts formed by SCLC cells in the CAM by Transcriptase-polymerase chain reaction (RT-PCR) and Western-blot analysis. This study investigated the effect of HIF-1α on the angiogenic potential of the SCLC cells at histological, morphological, and molecular levels. Furthermore, this MAPK inhibitor study demonstrated that HIF-1α may be used as a potential

target for the treatment of SCLC in the future. Methods Cell culture and transduction with Ad5-HIF-1α and Ad5-siHIF-1α The NCI-H446 cell line was obtained from the American Type Culture Collection (ATCC; CAS; cell bank of Shanghai Institutes for Biological Sciences) and was cultured in RPMI-1640 medium (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100-μg/ml kanamycin at 37°C in a humidified atmosphere containing 5% CO2 and 20% O2. The medium was routinely Fludarabine purchase changed 2 d to 3 d after seeding. Cells were detached with trypsin/EDTA (GibcoBRL, Paisley, UK) and were resuspended in a 1:1 solution of serum-free RPMI-1640 medium to a final concentration of approximately 5 × 105 cells/10 μl. The appropriate transduction conditions of adenovirus (lengthen of time and multiplicity of infection-MOI) should be cleared for the analysis of microarry and

PCR. The high transduction efficiency of Ad5 (a tumor-specific and replication-defective adenovirus used as the control vector) could reduce experimental error and resulted in differential expression levels of HIF-1α in Ad5-HIF-1α and Ad5-siHIF-1α treatment groups, which was favorable to investigate the effect of HIF-1α on the growth of

NCI-H446 cells. We infected the cells by Ad5 and Ad5-siRNA and further eliminated the effect of adenovirus vector and non-targeting control siRNA. Ad5-EGFP, Ad5-siRNA-EGFP, Ad5-HIF-1α-EGFP and Ad5-siHIF-1α-EGFP adenoviruses were obtained from the Viral-Gene Therapy Department of Shanghai Eastern Hepatobiliary Surgery Hospital [21, 22]. The sequences of the HIF-1α primers were as follows: upstream sequence (5′CTAGCTAGCTAGACCATG GAGGGCGGC’3) and downstream sequence (5′CGGGATCCTTATCAGTTAACTTGATC C’3). The sequences of the siHIF-1α primers were as follows: upstream sequence (5′TCGAG GAAGGAACCTGATGCTTTATTCAAGAGATAAAGCATCAGGTTCCTTCTTA’3) these and downstream sequence (5′CTAGTAAGAAGGAACCTGATGCTTTATCTCTTGAATAAA GCATCAGGTTCCTTCC’3). As for Ad5-siHIF-1α, the pSilencer adeno 1.0-CMV system was purchased from Ambion for adenovirus construction. According to the manufacturer protocol deno-siHIF-1α was packaged and produced as the adenoviral backbone plasmid and the shuttle vector containing the siRNA template were linearized with PacI and then recombined in HEK-293 cells. After 10 days, Ad-siHIF-1α was obtained [22]. For the transduction experiments, cells were cultured in 6-well plates and were exposed to viral supernatants in the absence of cytokines and serum with different MOI.