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.