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. 2016 Aug 19;7(39):63003–63019. doi: 10.18632/oncotarget.11416

Figure 5. Knockdown of RRM2 using siRNA impairs the viability of Ewing sarcoma cells.

Figure 5

A. Western blot showing levels of the RRM2 protein in A673 cells after transfection with two different siRNAs targeting RRM2 or a non-targeting siRNA (si_NT). B. Relative viability of A673 cells treated with a non-targeting siRNA or the two siRNAs that target RRM2. C., D. Increase in caspase-3/7 activation in Ewing sarcoma cells lines C. and non-Ewing sarcoma cell lines D. treated with siRRM2_R2B or si_NT for three days. Results are plotted as the fold increase in caspse-3/7 activity in the cells treated with siRRM2_R2B compared to the cells treated with si_NT. E. Western blot showing levels of the RRM2 protein after transfection with si_RRM2_pool or the individual siRNAs that compose the pool set. The relative expression level of RRM2 for each siRNA compared to the non-targeting siRNA is shown below the plot. F. Relative viability of A673 cells transfected with si_NT, si_RRM2_pool and the individual siRNAs that compose the pool (1-way ANOVA, Dunnett's post hoc test) G. Venn diagram demonstrates the overlap between genes deregulated by ciclopirox (pink) and si_RRM2_R2B (green). H. Western blot showing that the exogenous, doxycycline-inducible RRM2 gene, which is labeled with a 3X-FLAG tag, is resistant to knockdown by si_RRM2_3. I. Relative viability of EW8 cells with doxycycline-inducible expression of siRNA-resistant RRM2 after transfection with si_RRM2_3. Cell viability was assessed using the CellTiter-Glo Luminescent Assay. J. Increase in caspase-3/7 activation in EW8 cells with doxycycline-inducible expression of siRNA-resistant RRM2 after transfection with si_RRM2_3. Fold change is relative to the si_NT cells. Data B., C., D., F., I., J. represent mean ± SD, n = 3. *** p -value < 0.001, **** p -value < 0.0001.