CC inhibits NMD in human cells.

A. Schematic diagram of the dual color bioluminescence-based NMD reporter construct containing CBR-TCRβ(PTC) and CBG-TCRβ(WT) transcription units. B. Ratios of CBR to CBG bioluminescence signals in U2OS cells stably expressing a dual color bioluminescence-based NMD reporter (hereafter referred to as U2OS reporter cells). Cells were treated with indicated concentrations of CC, or caffeine for 24 hours before imaging. The CBR/CBG ratio of the DMSO alone control was normalized to 1. Data represent the mean ± SD of three independent experiments. ****p ≤ 0.0001; **p ≤ 0.01; *p ≤ 0.05 (paired t-test). C. Ratios of CBR to CBG bioluminescence signals in U2OS reporter cells treated with DMSO or CC (10 μM) for the indicated times. The CBR/CBG ratio of the 0-hour time point was normalized to 1. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01 (paired t-test). D. Ratios of CBR to CBG reporter mRNAs in U2OS reporter cells treated with DMSO or CC (10 μM) for 24 hours. The CBR/CBG mRNA ratio of the DMSO alone control was normalized to 1. Data represent the mean ± SD of three independent experiments. *p ≤ 0.05 (paired t-test). E. Western blot result of the NMD reporter proteins (HA-tagged) after 24-hour treatment of U2OS reporter cells with DMSO or CC (10 μM). F. Ratios of CBR to CBG bioluminescence signals in Calu-6 cells infected with adenoviruses expressing the NMD reporter after 24-hour treatment with DMSO or CC (10 μM). The CBR/CBG ratio of the DMSO alone control was normalized to 1. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01 (paired t-test). G. Ratios of CBR to CBG bioluminescence signals in BJ cells infected with adenoviruses expressing the NMD reporter after 24-hour treatment with DMSO or CC (10 μM). The CBR/CBG ratio of the DMSO alone control was normalized to 1. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01 (paired t-test).