FIGURE 3.

Knockdown of Pdcd4 increases the polysomal association of p53 mRNA. Cytoplasmic extracts of HepG2 cells transiently transfected with control or Pdcd4-specific siRNA were fractionated by sedimentation through 10–50% sucrose density gradients. Aliquots of the gradient fractions were analyzed by agarose gel electrophoresis to visualize the distribution of 18 S and 28 S ribosomal RNAs (upper). Subsequently, fractions 1–3, 4–7, and 8–11 were pooled as nonribosomal, monoribosomal, and polyribosomal fractions as indicated. Total RNA isolated from these pools was then analyzed by quantitative real-time PCR for p53 (black bars) and Mdm2 (white bars) mRNAs. The bar height reflects the amounts of p53 and Mdm2 RNAs normalized to the amount of the corresponding RNA in the nonribosomal pool.