Figure 2.

Non-AUG translation reporters are resistant to inhibition by CHX. (A) HeLa cells were transfected with the nLuc-3XFlag reporters followed by treatment with 100 µg/mL CHX and quantification of luminescence signals. (B) After 1 or 24 h of treatment with 100 µg/mL CHX, HeLa cells were subjected to puromycin (PURO) labeling and Western blotting (WB) to confirm global translation inhibition. Tubulin was used as a loading control. (C) Luminescence from the nLuc-3XFlag reporters was measured at the indicated time points after 100 µg/mL CHX treatment. Data for each reporter were set relative to the 0-h time point and are shown as mean ± SD. n = 3. (D) Western blotting using an anti-Flag antibody was used to examine expression of the nLuc-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-nLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (E) Luminescence from the destabilized nLuc-3XFlag-PEST reporters was measured at the indicated time points after CHX treatment. Data for each reporter were set relative to the 0-h time point and are shown as mean ± SD. n = 3. (F) HeLa cells were pretreated (15 min) with vehicle (Veh.; 0.1% DMSO) or 100 µg/mL CHX followed by transfection of the nLuc-3XFlag reporters. Luminescence was measured after 24 h. (G) Raw luciferase values of nLuc-3XFlag reporters 24 h after transfection. Data are shown as mean ± SD. n = 3. (H) Luminescence signals for each reporter were quantified and set relative to the associated vehicle-treated samples. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (I) Western blotting was used to examine expression of the FFLuc-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-FFLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (J) HeLa cells were transfected with expanded (CGG)₁₀₀ repeat RAN translation reporters for 24 h and then collected (control) or treated for 24 h with 100 µg/mL CHX. “+1” and “+2” refer to the reading frame of the (CGG)₁₀₀ repeat in relation to the nLuc-coding sequence. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3.