Fig. 2.

MYCN overexpression confers sensitivity to R9-caPep. SK-N-BE(2)c cells were transfected by a siRNA (siMYCN) targeting MYCN mRNA or a non-targeting control siRNA (siCTL). 48 h after the initial transfection, cells were reverse transfected again by the same siRNA. a) SK-N-BE(2)c cells transfected by siMYCN or siCTL were incubated in the presence of 20 μM R9-caPep for 12 h. Intracellular MYCN, γH2A.X, and total H2A.X levels were determined by Western blot. The density levels of γH2A.X and total H2A.X were quantified by TotalLab Quant (Tyne and Wear, England) and were presented at the bottom of the panel. b) After the second transfection, cells were seeded into a 96-well plate directly and allowed to attach overnight. The cells were then treated by various concentration of R9-caPep for 72 h, before their growth was measured by a CellTiter-Glo assay. The R9-caPep IC₅₀ values from three independently transfected cell populations were averaged and graphed ± standard deviation (S.D.). c) MYCN protein levels in cell lysates extracted from aliquots of cells at 0, 72, and 144 h after the first siRNA transfection were examined by Western blot.