Figure 5.

The biological effect of modified ASOs targeted to MDR1 mRNA in KB-8-5 cells

(A and B) Real-time analysis of the effect of modified ASOs on the growth rate of KB-8-5 cells in the presence of vinblastine. Cells were transfected with 1 μM of oligonucleotides R1, M1, or G2, or 0.1 μM of oligonucleotides R2, G4, or M3 pre-complexed with Lipofectamine 2000 for 4 h; LF, cells treated with Lipofectamine 2000 only. Cell viability was recorded in real time using an xCELLigence instrument for 96 h. Vinblastine was added to the cells to a concentration of 300 nM 24 h post-transfection, and the cells were cultured in an atmosphere of 5% CO₂ at 37°C for 96 h. The results are shown as mean cell index ± SE. The data were statistically processed using Student's t test (two-tailed, unpaired); a p value of ≤0.05 was considered to indicate a significant difference; ∗∗data were statistically insignificant. (C and D) Analysis of the effect of modified ASOs on the level of MDR1 gene expression. Data of qPCR. KB-8-5 cells were transfected with ASOs R1, M1, G2 (0.5 μM) or R2, M3, G4 (0.1 μM) pre-complexed with Lipofectamine for 4 h and incubated for 24 and 48 h. LF, cells treated with Lipofectamine 2000 only. The expression level of MDR1 mRNA was normalized to the level of HPRT1 mRNA. (E and F) Western blot analysis of Pgp 72 h after transfection. β-Actin served as an internal control. Cells were transfected with oligonucleotides R1 (0.5 μM), M1 (0.5 μM), M3 (0.1 μM), G4 (0.1 and 0.5 μM), and G2 (0.5 μM) pre-complexed with Lipofectamine 2000 for 4 h and incubated for 72 h; LF, cells treated with Lipofectamine 2000 only. (D) The bar graph shows the semi-quantitative analysis of the western blot results for Pgp. Data of qPCR and western blot were statistically processed using one-way ANOVA with the Tukey post hoc test; p < 0.05 was considered to be statistically significant; ∗∗data were statistically insignificant. The data are presented as the mean three independent experiments with triplicate samples ±SEM.