Data Availability StatementAll relevant data are within the manuscript. analog, CC does not impact the kinase activity of SMG1, an essential NMD element and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is definitely self-employed of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC like a NMD inhibitor, which has implications for its use in basic research and drug development. Introduction First found out in and (Fig 1B and 1C). This level of Nodinitib-1 inhibition is similar to that caused by treatment with caffeine (10 mM, 24 hrs), an inhibitor of SMG1 (Fig 1B)[17], or by shRNA-mediated knockdown of NMD factors such as SMG1, UPF1 and UPF2[19]. Open in a separate windowpane Fig 1 CC inhibits NMD in human being cells.A. Schematic diagram of the dual color bioluminescence-based NMD reporter create comprising CBR-TCR(PTC) and CBG-TCR(WT) transcription devices. B. Ratios of CBR to CBG bioluminescence signals in U2OS cells stably expressing a dual color bioluminescence-based Nodinitib-1 NMD reporter (hereafter referred to as U2OS reporter cells). Cells were treated with Nodinitib-1 indicated concentrations of CC, or caffeine for 24 hours before imaging. The CBR/CBG percentage of the DMSO only control was normalized to 1 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 M) for the indicated times. The CBR/CBG ratio of the 0-hour time point was normalized to 1 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 M) for 24 hours. The CBR/CBG mRNA ratio of the DMSO alone control Nodinitib-1 was normalized to 1 1. Data represent the mean SD of three independent experiments. Nodinitib-1 *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 M). 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 M). The CBR/CBG ratio of the DMSO alone control was normalized to 1 1. Data represent the mean LRCH1 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 M). The CBR/CBG ratio of the DMSO alone control was normalized to 1 1. Data represent the mean SD of three independent experiments. **p 0.01 (paired t-test). To confirm the results obtained from bioluminescence imaging, we measured CBR and CBG mRNA and protein levels using RT-qPCR and western blot, respectively. Consistent with the results of bioluminescence imaging, CC treatment increased the ratio of CBR-TCR(PTC) to CBG-TCR(WT) at both mRNA and protein levels (Fig 1D and 1E). Treating the human lung cancer cell line Calu-6 or non-transformed BJ human fibroblasts with CC also resulted in NMD inhibition as measured by the NMD reporter (Fig 1F and 1G), indicating that the effect of CC on NMD is not a cell line-specific phenomenon. To further validate that CC is a bona fide inhibitor of NMD, we determined its effects on the stability of the endogenous mutant p53 mRNA in Calu-6 cells, which contains a PTC mutation[34]. To do this, cells were first treated with CC for 24 hrs. Subsequently, the transcription inhibitor actinomycin.