TAQC substrates can be degraded by both the proteasome and lysosomes. (A) U2OS cells transfected with ssGFP were treated with the indicated inhibitors for 4 h and imaged. Arrowheads indicate nucleolar localization of SSGFP. Scale bars, 5 μm. (B–D) Quantification of GFP fluorescence in SSGFP-transfected cells treated with the indicated inhibitors. Shown is box and whiskers plot with all points indicated. The boxes show the upper quartile, the median, and the lower quartile. ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001 by one-way ANOVA’s Dunnett’s multiple comparison test. n > 20 cells in randomly selected fields from two independent experiments. (E)SSGFP accumulates in nucleoli in MG132-treated cells. U2OS cells expressing SSGFP were treated with MG132 and stained with anti-nucleolin antibodies (magenta). Scale bar, 10 μm. (F)SSGFP accumulates in LAMP1-mCherry (mCh)–positive lysosomes in Baf A1–treated cells. Shown are two time points from a live-cell imaging experiment. Scale bar, 8 μm. (G) Schematic illustration of the translation-stalled ER reporters and their degradation pathways. L: lysosome; P: proteasome. (H) Left panels, ERGFP_NS-transfected HEK293T cells were treated with Baf A1 (200 nM) or MG132 (20 µM) and imaged continuously for 6 h. Scale bars, 5 μm. The graph shows the quantification of the experiment. Error bars, SEM. n = 6 cells for each condition.