To study the expression of survivin induced by hypoxia, A549 cells were incubated in hypoxic condition (1% O2, 5% CO2 and 94% N2) for Crenolanib chemical structure 24 h. Immunohistochemistry Immunohistochemical staining using the streptavidin peroxidase method (S-P method) was performed on 4-μm sections of paraffin-embedded specimens to detect expression of survivin and HIF-1α protein in NSCLC and benign lung disease tissues. In brief, after deparaffinization and hydration, the slides were treated with endogenous peroxidase in 0.3% H2O2 for 30 min, after which the sections were blocked for 2 hrs at room temperature
with 1.5% blocking serum in phosphate-buffered saline (PBS). Sections were then incubated with anti-Survivin antibody (1:200 dilution) or anti-HIF-1α antibody (1:200 dilution) at 4°C overnight., followed by washing in PBS, and incubation with secondary find more anti-mouse biotinylated antibody (1 : 2000) in PBS for 30 min at 37°C. Antibody binding was detected using the streptavidin-biotin-peroxidase complex/HRP, Code K0377 (Dako), with 3,3 diaminobenzidine for 3 min as a chromogenic substrate. Finally, the slides were lightly counterstained with hematoxylin.
As a negative control, duplicate sections were immunostained without exposure to primary antibodies. The results were observed under a light microscope. PCR-based Site Directed Mutagenesis of survivin promoter Genomic DNA of A549 cells was extracted with Universal gene DNA extraction kit ver.3.0 according to the manufacturer’s instructions. Survivin core promoter 230 bp (-203 ~ +27 bp) was amplified by PCR using primers with
sequences selected from the survivin core promoter sequence; (Forward: 5′-ATC GAC GCG TTC TTT GAA AGC AGT CGA GGG GGC-3′, Reverse: 5′-CCC AAG CTT TCT GGC GGT TAA TGG CGC GCC-3′,). The almost cycling parameters were 95°C for 10s as a pre-denature step, followed by 40 cycles of 95°C for 5s, and 55°C for 30s, 72°C for 10 min. PCR products were purified, a polyadenylated by T4 DNA ligase, and then cloned to www.selleckchem.com/products/i-bet151-gsk1210151a.html T-vector, named pGEM-T-EASY-sur230 bp. The template for site-directed mutagenesis was pGEM-T-EASY-sur230 bp. The forward and reverse primers (Forward: 5′-AGC GCT CCC GAC ATG CCC CGC GGC-3′, Reverse: 5′-GCC CTCTTA GGC GGT CCA C-3′) were used for PCR amplification. The cycling parameters were 30 cycles of 95°C for 10s, 60°C for 5s, 72°C for 30s. The linear product was self ligated after a blunting kination reaction; the product was named pGEM-T-EASY-sur229 bp and confirmed by sequencing. Construction of survivin promoter-luciferase reporter vectors, and transfection into A549 cells The mutant, and normal constructs were removed from pGL3-basic by restriction endonuclease Mlu I/Hind III.