Number of ribosomes per FL RNA and elongation rate. (A and B) Representative images of a GFP/RFP cell expressing ΔNC (no puromycin) (A) and KIF1C after puromycin treatment (B) acquired on a LSM980 confocal microscope. Merge and MIP are presented. Red arrow indicates a free FL RNA molecule, yellow arrow indicates a brighter translating protein foci colocalized with the FL RNA, and green arrows indicate fully synthesized proteins. Scale bars are 10 and 1 µm for zoom insets. (C) Dot plot representing 400 randomly selected values of green fluorescence intensities for fully synthesized KIF1C (26,058 spots) and Gag translation sites (14,768 spots) proteins from 18 to 19 cells, respectively. (D) Histogram representing the frequency distribution of the number of ribosomes per FL RNA. Red arrow points the average. The number of ribosomes was determined from 5,605 spots corresponding to single polysomes, using the fluorescence intensity of calibrator, and corrected according to ribosome position (X). (E) Live cells analysis with the quantification of the fluorescence intensity of individual polysomes in function of time (n = 12 traces from five cells). Videos were acquired on a confocal spinning disk microscope at a rate of one stack every 10 s. Traces were normalized by the mean intensity of their plateau and aligned from the start of the fluorescence decrease. (F) Mean representation of traces in E ± SEM. ΔT was calculated from a linear fitting to y = 0 and starting at t + 90 s.