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. 2015 Feb 17;12(1):83–92. doi: 10.3892/mmr.2015.3368

Figure 2.

Figure 2

Transfection with Ad-SOCS3 upregulates the expression levels of SOCS3 in NCI-H446 cells and inhibits their proliferative and angiogenic potential. (A) Determination of transduction conditions and evaluation of SOCS3 induction efficiency. Five different MOIs (20, 30, 40, 50 and 70) were assessed in the transduction experiment (60 h). The transduction efficiency was the highest when the MOI was 50 (MOI 50 vs. MOI 40; P<0.05; MOI 50 vs. MOI 70; P<0.05). Transduction efficiency of NCI-H446 cells with Ad5-GFP after 60 h is shown in the fluorescent image (MOI 50; magnification, ×200). After the cells were transduced with Ad5 and Ad5-SOCS3 (MOI, 50), the mRNA expression levels of SOCS3 were measured using quantitative polymerase chain reaction (NCI-H446/Ad5-SOCS3 group vs. control group, P<0.01; NCI-H446/Ad5 group vs. NCI-H446/Ad5-SOCS3 group, P<0.01) (B) Western blot analysis of SOCS3 and VEGF-A protein expression. Representative images of three independent experiments (lane 1, SOCS3 and VEGF-A protein expression in the control group; lane 2, SOCS3 and VEGF-A protein expression in Ad5-SOCS3 transfection group; lane 3, SOCS3 and VEGF-A protein expression in Ad5 transfection group) and relative expression of SOCS3 and VEGF-A protein normalized to β-actin (SOCS3 expression in Ad5-SOCS3 transfection group vs. control group, P<0.05; SOCS3 expression in Ad5-SOCS3 transfection group vs. Ad5 transfection group, P<0.05; VEGF-A expression in Ad5-SOCS3 transfection group vs. control group, P<0.01; VEGF-A expression in Ad5-SOCS3 transfection group vs. Ad5 transfection group, P<0.01). (C) Growth curve of the cells in two groups. In the control group, NCI-H446 cells entered the period of logarithmic growth after the fourth day. Following transfection with Ad5-SOCS3, cell growth was significantly decreased and the logarithmic growth phase was not entered (NCI-H446/SOCS3 group vs. NCI-H446, P<0.05). Ad5, tumor-specific replication-defective adenovirus type 5; SOCS3, suppressor of cytokine signaling 3; VEGF, vascular endothelial growth factor; MOI, multiplicity of infection; GFP, green-fluorescent protein.