(A) Schematic depicting the SunTag translation reporter system used in this study, as described by Wu et al., 2016. Note that although the SunTag reporter construct harbors MS Coat Protein Binding Sites (MBSV5) in the 3’ UTR, we did not employ RFP-labeled MS Coat Protein for mRNA tracking. (B) Representative spinning disk confocal images (100x) of a live HEK-293FT cell before and 2 min after addition of 220 µM puromycin. Regions depicted in white dashed lines are displayed in the inset. Scale bars: 10 µm for large field, 2 µm for inset. (C) Live imaging time course of SunTag puncta in cells with pretreatments as indicated (355 µM cycloheximide or 54 µM emetine), followed by treatment as indicated (220 µM puromycin). Puromycin was added at 60 s into the 8 min imaging trial (dashed black line). SunTag puncta for each cell were normalized to the average SunTag puncta in that cell during the initial 60 s. Data are comprised of 3–4 replicate imaging trials per treatment condition, with each replicate containing 7–12 cells (Veh-Veh: n = 27 cells from three replicates, Veh-Puro: n = 30 cells from three replicates, Chx-Puro: n = 27 cells from three replicates, Emt-Puro: n = 41 cells from four replicates). Plotted are the mean ± standard deviation of all cells, computed in five second time intervals (see Materials and methods). (D) The mean normalized SunTag puncta in each replicate imaging trial was used to determine the time after puromycin addition at which two thirds, half, and one third of SunTag puncta remain in each condition. Welch’s t-test was used to assess differences between Veh-Puro and Emt-Puro (for 66.6%: t(5) = 3.932, p=0.0271, for 50%: t(5) = 3.183, p=0.0294, for 33.3%: t(5) = 0.967, p=0.3996). * denotes p<0.05.