Frequency changes of the three cliques after translation inhibition by rapamycin. (a, top left) Phosphorylation responses by immunoblotting after 1-, 6-, and 24-h treatment with 100 nM rapamycin of known mTORC1 downstream targets (4E-BP, RPS6, and p70-RPS6K) in MCF7 cells. (bottom left) The decrease of protein synthesis was measured by means of AHA incorporation using Click-iT AHA Alexa Fluor 488. The methionine analogue AHA in methionine-free DMEM was added to cells for 30 min. Cells were fixed and incubated with fluorescent alkyne to label the AHA incorporated into nascent proteins. The relative AHA incorporation was assessed by using Operetta HCS. Experiments were run in biological triplicate (mean ± SD is displayed), and 700–1,000 cells were considered for each sample. The kinetics of protein synthesis inhibition (open squares) and the corresponding decrease in polysome content (closed circles) are shown. (middle) Galleries of unclassified HMW polysomes before and after treatment of MCF7 with 100 nM rapamycin. (right) HMW polysomes purified from control cells and cells treated with 100 nM rapamycin for 6 h were adsorbed on the mica and the number of ribosomes in each polysome was counted. The corresponding distributions of the numbers of ribosomes per transcript are shown. (b) Comparison between galleries of classes of HMW polysomes before and after 100 nM rapamycin treatment for 6 h. After grouping the 2D rotational spectra images, the differences in rotational symmetry for distinguishing classes are shown (mean ± SD is displayed). The classification has been performed on 502 and 197 objects purified from MCF7 polysomes before and after rapamycin treatment. (a and b) Bars, 100 nm. (c) Frequencies of rectangular (blue), rounded (green), and triangular cliques (magenta) observed at different rapamycin concentrations are compared with the corresponding protein inhibition (open squares). Protein inhibition is relative to the control (no rapamycin) and was determined by AHA incorporation.