Figure 5.

Depletion of rpS6 upregulates 5´-TOP mediated translation. (a) Western blots showing levels of p53 and β-actin in A549 cells transfected with either rpS6 or rpL7a siRNA and treated for 6 h with the indicated doses of cycloheximide (CHX). (b) Measurement of [³⁵S]methionine incorporation in cells transfected with the indicated siRNAs and treated, or left untreated, for 4 h with 0.1 µg ml⁻¹ cycloheximide. Results are shown as means ± s.e.m. for three independent experiments. (c) Quantification of the ratio of p53 to β-actin (measured by densitometry on western blots) in cells transfected for 24 h with either rpS6 siRNA (open diamonds) or rpL7a siRNA (filled squares) and treated for the indicated times with 0.1 µg ml⁻¹ cycloheximide. Results are shown as means ± s.e.m. for three independent experiments. A representative western blot used for this analysis is shown in Supplementary Information, Fig. S4c. (d) Polysome profiles (top) and northern blot analysis of the distribution of rpL11 mRNA on polysomes (bottom) in HEK 293 cells co-transfected with wild-type (WT) rpS16-hGH plasmid DNA in combination with either NSsiRNA (black line) or rpS6 siRNA (grey line). (e) Northern blot analysis of the distribution on polysomes of hGH reporter mRNA in HEK 293 cells co-transfected with NSsiRNA in combination with either wild-type rpS16-hGH or Cm5rpS16-hGH plasmid DNA. (f) Northern blot analysis of the distribution on polysomes of hGH reporter mRNA in HEK 293 cells co-transfected with rpS6 siRNA2 in combination with either wild-type rpS16-hGH or Cm5rpS16-hGH plasmid DNA. In the northern blot analysis of all three panels, fractions 4–12 contained translating polysomes. (g) Model of p53 stabilization in response to impaired 40S ribosome biogenesis. See the text for details. NPL, nucleoplasm; NO, nucleolus. Uncropped images of blots are shown in Supplementary Information, Fig. S5.