Figure 5

Changes in Dox-induced cytotoxicity after the downregulation of miR-125b and Bak in p53-truncated EWS cells. A) miR-125b was stably knocked down in SK-N-MC cells which express a truncated mutant of p53. The cells were seeded at 2 × 10³ cells/well in 96 well plates and cultured for 12 h, then were treated with various concentrations of doxorubicin for an additional 48 h. (Left) The cell viability was determined by the CellTiter-GloTM Luminescent Cell Viability Assay. The data represent the means of three separate experiments. The results are the means ± SD. *, P < 0.05. (Right) The whole cell lysates were subjected to an immunoblot analysis using antibodies against Bak and β-actin. B) SK-N-MC cells were transfected with 20 nM of siRNA against Bak. (Upper) After 48 h incubation, the cells were collected, and qRT-PCR and an immunoblot analysis were performed to confirm the effect of the siRNA. (Lower) Cells were seeded at 2 × 10³ cells/well in 96 well plates, and were treated with various concentrations of doxorubicin for 48 h. The cell viability was determined by the CellTiter-GloTM Luminescent Cell Viability Assay. The data represent the means of three separate experiments. The results are the means ± SD. *, P < 0.05. C) p53, Bak or mock construct was introduced into SK-N-MC cells using a lentivirus. After 96 h incubation, the cells were seeded at 2 × 10³ cells/well in 96 well plates. Twelve hours later, the cells were treated with various concentrations of doxorubicin for an additional 48 h. The cell viability was detected by the CellTiter-GloTM Luminescent Cell Viability Assay. The data represent the means of three separate experiments. The results are the means ± SD. *, P < 0.05. Immunoblot analyses were performed to confirm the effects of the lentivirus induction.