Figure 1

A. Up-regulation of PUMA at the transcription level after Ad-E2F-1 expression in SK-MEL-2 cells. SK-MEL-2 cells were infected with Ad-E2F-1 at MOI 100. Ad-LacZ was used as control. Cells were harvested after infection at 8 hours, 12 hours, and 16 hours. Real-time PCR was performed as described in Materials and Methods. β-actin was used as an internal control. The bar graphs are expressed as fold increase relative to Ad-LacZ-infected cells adjusted for β-actin. Each of the experiments is a representation of two independent experiments performed in duplicate. After adjustment for β-actin mRNA expression, real-time PCR results showed a 2.6-, 5-, and 10-fold induction at the PUMA mRNA level after 8 hours, 12 hours and 16 hours of Ad-E2F-1 infection, respectively. B. Up-regulation of PUMA at the protein level after Ad-E2F-1 expression in SK-MEL-2 cells. Western blot analysis of PUMA at indicated time points after Ad-LacZ or Ad-E2F-1 infection. Anti-actin was used to show the equal loading and transfer in each lane. E2F-1 expression was also shown at the indicated time points to correlate with PUMA expression. PUMA/actin showed the ratio of the scanned optical density of each protein band. In SK-MEL-2 cells, PUMA protein level increased at 8 hours post-infection with Ad-E2F-1 and was still pronounced at 16 hours and 18 hours post-infection with Ad-E2F-1.