Cytometry was used to calculate the cell number and the efficienc

Cytometry was used to calculate the cell number and the efficiency of transduction was estimated by determining the percentage of enhanced green fluorescence protein (EGFP)-positive cells. The appropriate MOI was chosed using the following formula: MOI = titer (pfu) × viral fluid (L)/cell number. When the MOI was 50, the transduction efficiency was more than 95% and expression was stable in a transduction experiment for 60 h (Figures 1A and 1B). In order to eliminated the effect of empty vector Ad5 and non-targeting control siRNA: Ad5-siRNA on HIF-1α mRNA expression and SCLC

cells growth, transduction of NCI-H446 cells with Ad5 and Ad5-siRNA were carried out. In five selected time stages we found that empty vector Ad5 and Ad5-siRNA had no significant effect on the HIF-1α mRNA expression(Figure TPCA-1 purchase 1C). We selected the group(MOI = 50) for the high and stable transduction efficiency in the following experiments. HIF-1α mRNA levels in the NCI-H446 cells KU55933 nmr were measured by real-time PCR in our laboratory. The expression of HIF-1α mRNA was the highest in the Ad5-HIF-1α -treated cells and lowest in the Ad5-siHIF-1α-treated cells 60 h after transduction (Figure 1D). In addition, exogenous HIF-1α transduction significantly induced NCI-H446 cells growth and empty vector Ad5 and Ad5-siRNA transduction had no significant effect on the growth of NCI-H446 cells (Figure 1E). Figure 1 Transduction of NCI-H446 cells with Ad5. Chosing

transduction condition and the effect on NCI-H446 cells growth by HIF-1α. (A)Five different multiplicities of infection (MOI: 20, 30, 40, 50, and 70) were tested in the transduction experiment (60 h). The transduction efficiency was the highest when the MOI was 50 (*p < 0.05 represents MOI50 vs. MOI40; **p < 0.05 represents MOI50 vs. MOI70). (B) Transduction efficiency of NCI-H446 cells with Ad5-EGFP after 60 h (MOI = 50; 200 ×). (C) After the cells were transduced with Ad5 and

Ad5-siRNA(MOI = 50), the mRNA expression level of HIF-1α was measured in the indicated time Verubecestat period by real-time Bcl-w PCR (*p > 0.05 represents NCI-H446/Ad5 group vs control group; ▲p > 0.05 represents NCI-H446/Ad5- siRNA group vs control group;) (D)After the cells were transduced with Ad5-HIF-1α and Ad5-siHIF-1α (MOI = 50), the mRNA expression level of HIF-1α was measured in the indicated time period by real-time PCR (*p < 0.05 represents NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group, 60 h vs. 48 h; ** p < 0.05 represents NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group, 60 h vs. 72 h). (E) Growth curve of the cells in five groups. After transduction with Ad5 and Ad5-siRNA, the trendency of growth curve had no significant change. After transduction with HIF-1α, the growth curve of NCI-H446 cells shifted to the left with the growth of cells entering the period of logarithmic growth. After transduction with Ad5-siHIF-1α, however, the growth curve shifted to the right (*p > 0.

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