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To inhibit nonsense-mediated decay (NMD) in HEK293T cells using cycloheximide (CHX), a concentration of 100 µg/mL is commonly used to effectively inhibit translation without significant toxicity [1][2]. Optimal exposure time ranges from 4-6 hours, balancing NMD inhibition and maintaining cell viability [1][2]. Shorter exposure times (2-4 hours) can also be effective if toxicity is a concern [1]. However, conditions may vary based on specific experimental setups and cell line responses. Monitoring cell health and performing optimization experiments are crucial for achieving the best results [3][4].

Reference

[1] Li, Z., Vuong, J. K., Zhang, M., Stork, C., & Zheng, S. (2017). Inhibition of nonsense-mediated RNA decay by ER stress. RNA, 23, 378 - 394.

[2] Wang, D., Zavadil, J., Martin, L., Parisi, F., Friedman, E., Levy, D., Harding, H., Ron, D., & Gardner, L. B. (2011). Inhibition of Nonsense-Mediated RNA Decay by the Tumor Microenvironment Promotes Tumorigenesis. Molecular and Cellular Biology, 31, 3670 - 3680.

[3] Wengrod, J., Martin, L., Wang, D., Frischmeyer-Guerrerio, P., Dietz, H., & Gardner, L. B. (2013). Inhibition of Nonsense-Mediated RNA Decay Activates Autophagy. Molecular and Cellular Biology, 33, 2128 - 2135.

[4] Soares, R. O., Connelly, Z., Vázquez, O., & Carastro, L. (2014). Optimization of Chemical Inhibition Conditions for NMD Studies in p53‐Positive Cancer Cell Lines (LB234). The FASEB Journal, 28.