Figure 2. Probing translation sites with translation inhibitors.
(A) Images of translation foci in the same field of view before and after treatment of 275 μM puromycin (Puro) at 37 °C. Insets show the zoom-in of the boxed region. Scale Bars: 5 μm. The full time course is shown in Movie S1.
(B) Time courses of the normalized total intensities of the GFP foci in individual cells under different drug treatments. Thin black line: upon 275 μM Puro treatment. Thick black line: upon 200 μM Cycloheximide (CHX) treatment. Medium black line: upon 275 μM Puro treatment in the presence of 50 μM CHX. The curves are normalized to the average of the first 5 data points. Puro was added at the time indicated by the dashed line. The translation signal shows a decay after the Puro or Puro + CHX treatment. A single-exponential fit of the decay region for Puro treatment (red line) and for Puro + CHX treatment (purple line) gives the observed rate constant (kobs) of the foci disappearance after drug treatment.
(C) Plot of kobs vs. [Puro] under conditions of Puro treatment only (red) or Puro treatment in the presence of three different concentrations of CHX (purple, green and blue). Error bars are S.E.M. (n = 5–10 cells for each condition, in each cell the number of polysomes before drug treatment is 30 -100)
See also Figures S2, S3 and Movie S1.