Figure 2. SiRNA-based silencing of c-MYC.

Two different siRNAs targeting exon 2 and exon 3 of the human c-MYC sequence (NM_002467) were used. (A) A2780CP20 cells (2×10⁵) were transfected with 200 nM c-MYC-siRNA. Total protein was isolated from siRNA-transfected cells for Western blot analysis as described in the “Materials and Methods” section. Densitometric analysis of the intensities of the bands was calculated relative to the C-siRNA. Averages ±SEM are shown (****P<0.0001). (B) A2780CP20 cells (2×10³) were seeded into 96-well plates and 24-hr later cells were transfected with a serial dilution of C-siRNA or c-MYC-targeted siRNAs. Cell viability was calculated 72-hr post-transfection as described in the “Materials and Methods” section. Percentages were obtained after blank OD subtraction, taking the untreated cells values as a normalization control. Averages ±SEM are shown. (C) A2780CP20 cells (6×10⁴) were seeded into 6-well plates and 24-hr later 100 nM c-MYC-siRNA(2) or 100 nM C-siRNA was added to the cells. Eight hours post-transfection, 1000 cells were seeded into 10-cm Petri dishes. Seven days later, cells were stained and colonies of at least 50 cells were scored under a light microscope. The % of clonogenicity was calculated relative to C-siRNA. Averages ±SEM are shown for three independent experiments (***P<0.001). (D) A2780 cells (3×10³) were seeded into 96-well plates and 24-hr later cells were transfected with a serial dilution of C-siRNA or c-MYC-siRNA(2). Cell viability was calculated as described in Figure 2B. Averages ±SEM are shown. (E) A2780CP20 cells (2×10⁵) were transfected with 50 nM c-MYC-siRNA(2). Total protein was isolated from siRNA-transfected cells for Western blot analysis as described in the “Materials and Methods” section. Densitometric analysis of the intensities of the bands was calculated relative to the C-siRNA. Averages ±SEM are shown (****P<0.0001). (F) A2780CP20 cells (2×10³) were seeded into 96-well plates and 24-hr later cells were transfected with 50 nM C-siRNA or 50 nM c-MYC-siRNA(2). The next day, the media was replaced by CIS (2 μM final concentration)-containing RPMI-1640 media. Cell viability was calculated 72-hr after CIS treatment (96-hr post-transfection) as previously described. Averages ± SEM are shown (*P<0.05, **P<0.01, ***P<0.001).