FIGURE 6.
The effect of proteasome inhibition on p53 promoter occupancy and p21waf1 RNA transcription. A, H460 cells were transfected with nonspecific siRNA control (n.c.) or Rpt6-specific siRNA duplexes, and treated with doxorubicin (+) or not (−) at 40 h after transfection. After an additional 8 h, cells were harvested or processed for ChIP and RNA expression analysis. Western blots showed ∼90% knockdown of Rpt6 protein expression both in the absence (−) and presence (+) of Dox induction. B, ChIP analysis of p53 occupancy on the p21waf1 promoter with and without Dox induction. Negative control and specific siRNA of Rpt6 are indicated in each lane. Real-time PCR values were normalized to the input (input values represent 1% of the total lysate), and data are presented as % input. C, real-time RT-PCR analysis of p21waf1 expression with and without Dox induction. Real-time PCR values were normalized to the glyceraldehyde-3-phosphate dehydrogenase control and calibrated to the negative control siRNA and without Dox induction samples. Data were presented as -fold increase of p21waf1 gene expression. D, the effect of MG132 treatment on p53 occupancy on the p21waf1 promoter. H460 cells were induced by Dox (+) or not (−) when cells were grown to ∼70% confluency. After 8 h, cells were treated with MG132 (+) (5 μm) or not (−) and grown for an additional 4 h. After that, cells were harvested or processed for ChIP, and data were analyzed in the same way above. E, the effect of MG132 treatment on p21waf1 expression. Real-time RT-PCR analysis of p21waf1 expression was done in the same way above.