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. 2018 Oct 5;13(10):e0204978. doi: 10.1371/journal.pone.0204978

Fig 2. CC stabilizes endogenous NMD targets.

Fig 2

A. Effects of CC on the stability of p53 mRNA containing a PTC in Calu-6 cells. Cells were treated with DMSO or CC (10 μM) for 24 hours followed by actinomycin D treatment for 6 hours to inhibit transcription. Total mRNA was collected before and after actinomycin D treatment. p53 mRNA levels were measured using RT-qPCR. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01 (paired t-test). B. Effects of CC on the levels of the SC35 mRNA isoforms (SC35C, SC35D, and SC35WT). U2OS cells were treated with DMSO or CC (10 μM) for 24 hours followed by total RNA isolation and RT-qPCR. The mRNA levels of DMSO-treated cells were normalized to 1. Data represent the mean ± SD of three independent experiments. *p ≤ 0.05; ns, not significant (paired t-test). C. Effects of CC on the stability of known NMD targets PIM3, UPP1, FRS2, and PISD in Calu-6 cells. Samples were collected and analyzed as depicted in (A). ORCL and HPRT are non-NMD target controls. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01; ns, not significant (paired t-test). D. Effects of CC on the stability of the mRNA of NMD factors UPF1, UPF2, SMG1, SMG5, SMG6, SMG7, and UPF3B in Calu-6 cells. UPF3B is not a NMD target. Samples were collected and analyzed as depicted in (A). Data represent the mean ± SD of three independent experiments. ****p ≤ 0.0001; **p ≤ 0.01; *p ≤ 0.05, ns, not significant (paired t-test). E. Effects of CC on the steady-state levels of the mRNA of NMD factors UPF1, UPF2, SMG1, SMG5, SMG6, SMG7, and UPF3B. Calu-6 cells were treated with DMSO or CC (10 μM) for 24 hours followed by total RNA isolation and RT-qPCR. Results represent fold change of mRNA levels in CC-treated cells, compared to DMSO-treated cells. ***p ≤ 0.001; **p ≤ 0.01; ns, not significant (paired t-test).