(A) Schematic representation of the live-cell translation reporter. An inducible degron (DHFR-Y100I) fused to sfGFP and an NLS is separated from an NLS-mCherry protein by a P2A ribosome skipping sequence, which allows these two proteins to be synthesized as separate proteins from a single transcript. Upon addition of the small molecule stabilizer trimethoprim (TMP), newly synthesized DHFR-sfGFP-NLS is stabilized and GFP fluorescence increases over time due to new GFP protein synthesis. Thus, GFP fluorescence increase reports on translation efficiency. The mCherry signal is used to normalize for the plasmid copy number per cell. (B) RPE-1 cells stably expressing the reporter were treated with 50 μM TMP and followed by time-lapse microscopy. Scale bar, 20 μm. Time is indicated in min. (C) Quantification of GFP/Cherry ratio (mean and standard deviation, n = 8 cells) with or without cycloheximide treatment. (D) 5′ and 3′ UTRs from indicated genes were inserted in the reporter. Cells expressing the different reporters were blocked in mitosis with taxol, treated with TMP and imaged for 4 hr. To determine the translation rate, the GFP/mCherry ratio was calculated at the start and end of each video for both interphase and mitotic cells. The ratio of translation rates in mitosis and interphase for each reporter is shown. For Emi1, mitotic cells were compared with G2 phase cells only, as translation was also reduced in G1 (unpublished observation). Results are mean and SEM of 3 independent experiments with 10–20 cells analyzed per condition per experiment.