Compound C inhibits nonsense-mediated RNA decay independently of AMPK

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor 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 independent 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 as a NMD inhibitor, which has implications for its use in basic research and drug development.


Introduction
First discovered in S. cerevisiae, nonsense mediated mRNA decay (NMD) is an evolutionarily conserved RNA quality control pathway that targets aberrant RNAs with PTCs for degradation [1]. Translation of PTC-containing mRNAs generates aberrant protein products, which may have pathological effects on the cell. Therefore, NMD plays an important protective role PLOS  ability of CC to induce autophagy is AMPK-independent [30]. Interestingly, disruption of NMD activity also results in activation of autophagy, which is, in part, due to the stabilization of the transcripts of ATF4, a transcription factor that promotes expression of multiple autophagy genes including LC3B and ATG5 [33]. These observations prompted us to test the possibility that CC inhibits NMD, which in turn leads to autophagy induction. By examining the effects of CC on a highly effective NMD reporter as well as endogenous NMD target transcripts, we demonstrate that CC indeed possesses a previously unrecognized activity in inhibiting NMD in human cells. The ability of CC to suppress NMD is not mediated through the inhibition of AMPK or SMG1, the only known kinase in the NMD pathway, but it down-regulates protein levels of several core NMD factors. Although CC treatment causes upregulation and stabilization of the NMD target ATF4, this effect is apparently not responsible for the induction of autophagy by CC.

CC inhibits NMD activity in human cells
To determine whether CC can modulate NMD efficiency in human cells, we used a previously-developed bioluminescence-based reporter system, which can accurately measure NMD activity in mammalian cells using multiple assays [19]. This reporter contains two transcription units that express a PTC-containing TCRβ minigene fused to CBR luciferase (CBR-TCRβ (PTC)) and a wild type TCRβ minigene fused to CBG luciferase (CBG-TCRβ(WT)), respectively ( Fig 1A). NMD activity is measured as the ratio of CBR-TCRβ(PTC) to CBG-TCRβ (WT) at the levels of mRNA, protein, or luciferase activity, with an increase in the ratio representing NMD inhibition [19]. To determine the effects of CC on NMD, we treated human U2 osteosarcoma (U2OS) cells stably expressing the reporter with CC and then measured NMD activity by bioluminescence imaging. CC inhibited NMD in a dose-and time-dependent manner, with~3-fold inhibition of NMD of the reporter observed after 24-hr treatment with CC at 10 μM, a concentration commonly used to inhibit AMPK in vivo and in vitro (Fig 1B and  1C). This level of 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].
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-TCRβ(PTC) to CBG-TCRβ(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 D was added to block new mRNA synthesis for 6 hrs. RT-qPCR was then performed to measure the levels of the p53 mutant mRNA immediately before and after actinomycin D treatment. As shown in Fig 2A, CC stabilized the PTC-containing p53 mRNA, further supporting the idea that CC inhibits NMD.

CC stabilizes physiological NMD targets
In addition to eliminating mutant mRNAs, NMD also regulates gene expression by targeting many non-mutant physiological transcripts for degradation [35]. To further illustrate the 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. ÃÃÃÃ p 0.0001; ÃÃ p 0.01; Ã 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. ÃÃ 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. Ã 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. ÃÃ p 0.01 (paired t-test). G. Ratios effects of CC on NMD, we determined whether CC can also stabilize physiological NMD targets. The SC35 (SRSF2) gene encodes three splicing variants, two of which (SC35C and SC35D) are targets of NMD [36]. We found that CC treatment increased the levels of SC35C and SC35D, but had no effect on SC35WT that is not targeted by NMD (Fig 2B), consistent with the results obtained from the NMD reporter described above. In further support of the idea that CC inhibits NMD, CC also stabilized endogenous NMD targets such as PIM3, UPP1, FRS2, and PISD, but had no effect on ORCL and HPRT, which are not NMD targets ( Fig 2C) [3,37]. Previous studies have shown that transcripts of several NMD factors including SMG1, UPF1, UPF2, SMG5, SMG6, and SMG7 are themselves targets of NMD, a feature that allows for autoregulation of the NMD pathway [38,39]. We predicted that inhibition of NMD by CC would cause stabilization of these transcripts. Indeed, CC stabilized the mRNA of these NMD factors ( Fig 2D). CC treatment also increased the steady-state levels of these transcripts except UPF2 (Fig 2E). UPF3B mRNA, which is not a NMD target, was not stabilized by CC, although its steady-state level increased (Fig 2D), suggesting that CC exerted additional effects on NMD factor transcripts. Taken together, these data strongly suggest that CC has a previously unrecognized activity in inhibiting NMD in human cells.

CC inhibits NMD independent of AMPK
CC has been widely used as an inhibitor of AMPK, although it also targets several other kinases [23]. To determine whether CC inhibits NMD through AMPK, we used siRNAs to knock down both isoforms of the catalytic subunit of AMPK (AMPKα1 and AMPKα2) in U2OS cells. As expected, AMPK depletion reduced the phosphorylation of acetyl-CoA carboxylase1 (ACC1) at serine 79, a direct AMPK phosphorylation site ( Fig 3A)[40]. However, AMPK knockdown did not influence the inhibitory effects of CC on NMD, suggesting that CC inhibits NMD independently of AMPK ( Fig 3B). To rule out the possibility that the residual AMPK present in the knockdown cells was sufficient to mediate CC's effects on NMD, we generated AMPKα-KO cells lacking both AMPKα1 and AMPKα2 using the CRISRP/Cas9 technology ( Fig 3C). No significant differences in the extent of NMD inhibition were observed between WT and AMPKα-KO cells after CC treatment (Fig 3D), indicating that AMPK inhibition is not responsible for CC's effect on NMD. Interestingly, we found that forced activation of AMPK using 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) did not enhance, but actually attenuated NMD activity (Fig 3E and 3F). These results strongly suggest that CC inhibits NMD independently of AMPK activity and that activation, but not inhibition, of AMPK suppresses NMD.

CC does not inhibit the kinase activity of SMG1, but it reduces the protein levels of multiple NMD factors
As a competitive ATP analog[21], CC is known to inhibit several other kinases in addition to AMPK[23]. One possible mechanism by which CC inhibits NMD involves the inhibition of SMG1, which is the only known protein kinase that directly phosphorylates the RNA helicase UPF1 to promote NMD [34]. To test this possibility, we performed an in vitro kinase assay to determine whether CC can inhibit the kinase activity of SMG1. We immunoprecipitated Histagged wild type (WT) or a kinase dead (DA) mutant of SMG1 expressed in 293T cells [34]. A purified recombinant GST-p53N fusion protein containing a N-terminal segment of p53 was 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. ÃÃ p 0.01 (paired t-test).  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. ÃÃ 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. Ã 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. ÃÃ p 0.01; ns, not significant (paired ttest). 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. ÃÃÃÃ p 0.0001; ÃÃ p 0.01; Ã 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. ÃÃÃ p 0.001; ÃÃ p 0.01; ns, not significant (paired t-test). used as a model substrate for SMG1 ( Fig 4A) [41]. As shown before, SMG1(WT), but not SMG1 (DA), efficiently phosphorylated GST-p53N ( Fig 4B) [41]. This kinase activity was abolished by caffeine, a known inhibitor of SMG1[17]. However, SMG1 kinase activity was not affected by CC at 10 μM, a concentration that efficiently inhibited NMD in cells (Figs 4B and 1). This result suggests that the inhibitory effects of CC on NMD do not result from SMG1 inhibition.
We next asked whether CC affected the protein levels of NMD factors, which would impact NMD activity. Interestingly, we found that CC markedly reduced the protein levels of 3 core

CC inhibits NMD independently of AMPK inhibition.
A. Effects of AMPKα-knockdown on ACC1 phosphorylation in U2OS reporter cells. Protein samples were collected 48 hours after siRNA transfection. B. Relative ratios of CBR to CBG bioluminescence signals in AMPKα-knockdown or control-knockdown U2OS reporter cells after 24-hour treatment with 10 μM CC or DMSO. The CBR/CBG ratio of the siControl DMSO control was normalized to 1. Data represent the mean ± SD of three independent experiments. Ã p 0.05; ns, not significant (paired t-test). C. Knockout (KO) of AMPKα in U2OS cells. D. Relative ratios of CBR to CBG bioluminescence signals in AMPKα-KO or control cells after 24-hour treatment with 10 μM CC or DMSO and infection with adenoviruses expressing the NMD reporter. The CBR/CBG ratio of the AMPK WT DMSO control was normalized to 1. Data represent the mean ± SD of three independent experiments. Ã p 0.05; ns, not significant (paired t-test). E. Effects of AICAR on ACC1 phosphorylation. Activation of AMPKα by AICAR also caused a gel mobility shift. F. Relative ratios of CBR to CBG bioluminescence signals in U2OS reporter cells after treatment with indicated concentrations of AICAR. The CBR/CBG ratio of untreated control was normalized to 1. Data represent the mean ± SD of three independent experiments. ÃÃ p 0.01; ns, not significant (paired t-test).
https://doi.org/10.1371/journal.pone.0204978.g003 NMD factors including UPF1, SMG5, and SMG6 (Fig 4C and 4D). CC treatment did not affect the protein levels of SMG1, UPF2, SMG7 and UPF3B, indicating the effects of CC are specific for a subset of NMD factors (Fig 4C and 4D). This downregulation of the protein levels of multiple NMD factors may in part underlie the inhibitory effects of CC on NMD.

CC augments the expression and stability of the NMD target ATF4, but ATF4 is dispensable for autophagy induced by CC
Our investigation of the effects of CC on NMD was initially inspired by the observations that both CC treatment and NMD disruption induce autophagy [30,33]. The autophagy-inducing with GST-p53 as substrate containing the phosphorylation site S15. CC (10 μM) was used to test effects on SMG1 kinase activity. Caffeine (10 mM), a known inhibitor of SMG1, was used as a positive control. C. Effects of CC on the protein levels of NMD factors UPF1, UPF2, UPF3B, SMG1, SMG5, SMG6, and SMG7 in U2OS reporter cells. D. Quantification of the effects of CC on the protein levels of NMD factors UPF1, UPF2, UPF3B, SMG1, SMG5, SMG6, SMG7. Quantification was performed by measuring signal intensity relative to actin. Protein levels of DMSO-treated cells are normalized to 1. Data represent the mean ± SD of three independent experiments. ÃÃ p 0.01; Ã p 0.05; ns, not significant (paired t-test). activity of CC in cells is unexpected as AMPK, which CC inhibits, has been shown to promote autophagy [42]. To investigate the relationships between CC, NMD, and autophagy, we first determined whether CC could induce autophagy under our experimental conditions. U2OS cells were treated with 10 μM CC and the induction of autophagy was assessed. Consistent with published findings, both western blot and immunofluorescence staining results show a dramatic increase in the levels of LC3B-II, a surrogate marker of autophagosome formation, after CC treatment (Fig 5A and 5B) [43]. Using transmission electron microscopy (TEM), we also detected a marked increase in autophagosome formation after CC treatment (Fig 5C and  5D). Knockdown of the NMD factor SMG1 or UPF1 also resulted in accumulation of LC3B-II, in agreement with previous observations (Fig 5E)[33]. These data indicate that both CC and NMD disruption do indeed induce autophagy. Given the inhibitory effects of CC on NMD activity described above, these observations suggest the possibility that NMD inhibition is part of the mechanism for the autophagy-inducing activity of CC.
It has been shown that the induction of autophagy in response to NMD inhibition is in part mediated by ATF4, a direct NMD target [33]. Upon NMD inhibition, the stabilization of ATF4 mRNA leads to increased ATF4 protein levels, which promote autophagy by activating the expression of autophagy genes including LC3B and ATG5 [44]. To determine whether ATF4 contributes to CC-induced autophagy, we first examined whether CC treatment causes stabilization and increase of ATF4 mRNA. RT-qPCR results indicate that CC treatment indeed increased both the stability and the steady-state levels of ATF4 mRNA (Fig 5F and 5G), leading to a dramatic increase in ATF4 protein levels ( Fig 5H). To determine whether this ATF4 upregulation is important for autophagy induction after CC treatment, we knocked down ATF4 using siRNA in cells and then examined LC3B-II accumulation after CC treatment. Depletion of ATF4 expression did not significantly abrogate LC3B-II accumulation (Fig 5I). To rule out the possibility that the residual ATF4 protein in the knockdown cells is sufficient for autophagy activation, we generated ATF4-knockout cells using CRISPR/Cas9 (Fig 5J). No significant differences were observed between parental cells and ATF4-knockout cells in LC3B-II accumulation after CC treatment, further supporting the idea that ATF4 is dispensable for autophagy activated by CC (Fig 5K).

Discussion
This study identifies CC as an inhibitor of NMD activity in human cells (Figs 1 and 2). Using both knockdown and knockout approaches, we show that the effect of CC on NMD is not mediated through the inhibition of the key metabolic regulator AMPK, adding another important "off-target" activity to CC (Fig 3A-3D)[23, 28, 45]. Interestingly, activation of AMPK by AICAR causes NMD attenuation (Fig 3E and 3F), raising the possibility that cellular metabolic state can influence NMD activity. In addition to AMPK, CC has been shown to inhibit several other protein kinases including ALK2, ALK3, ALK6, ERK8, MNK1, PHK, MELK, DYRK, HIPK2, Src, and Lck [22,23]. SMG1 is the only protein kinase among the identified NMD factors and thus could be a potential target of CC. However, CC did not inhibit the kinase activity of SMG1 in vitro at the concentration that efficiently abrogates NMD in cells (Fig 4B). Interestingly, we found that CC treatment reduced the protein levels of three core NMD factors including UPF1, SMG5, and SMG6 (Fig 4C and 4D). The downregulation of these NMD factors may in part underlie the repression of NMD by CC. The mRNA levels of UPF1, SMG1, SMG5, SMG6 and SMG7, however, were higher in CC-treated cells (which was at least in part caused by the stabilization of these transcripts as a result of NMD inhibition) (Fig 2E)[38, 39,46]. These observations raise the possibility that CC affects the mRNA translation or the Effects of CC on LC3BII levels in U2OS cells. Cells were treated with DMSO or CC (10 μM) for 24 hours and collected at the indicated time points. B. Immunofluorescence detection of LC3B foci in U2OS cells after 24-hour treatment with DMSO or CC (10 μM). DAPI was used to visualize nuclei. C. Representative electron microscopy images of U2OS cells after 24-hour treatment of with DMSO or CC (10 μM). The arrow marks an electron-dense autophagosome. N, nucleus. D. Quantification of autophagosomes formed after 24-hour treatment with DMSO or CC (10 μM). Two sections were made from 2 different blocks for each sample. Each section is 80 nm thick and 1.75 mm long. The number of autophagosomes per cell from the two sections was counted for each sample. In total, 89 cells were counted in DMSO control, and 88 cells were counted in the CC-treated sample. E. Effects of shRNA-mediated knockdown of UPF1 or SMG1 on LC3BII levels in U2OS cells, compared to the effects in U2OS cells treated with DMSO or CC (10 μM) for 24 hours. F. Effects of CC on ATF4 mRNA expression in U2OS cells treated with DMSO or CC (10 μM) for 24 hours. mRNA expression of DMSO-treated cells was normalized to 1. Data represent the mean ± SD of three independent experiments. ÃÃ p 0.01 (paired t-test). G. Effects of CC on ATF4 mRNA stability in U2OS cells. Cells were treated with DMSO or CC (10 μM) for 24 hours followed by treatment with actinomycin D for 6 hours. Total RNA was collected before and after actinomycin D treatment. ATF4 mRNA levels was measured by RT-qPCR. Data represent the mean ± SD of three independent experiments. Ã p 0.05 (paired t-test). H. ATF4 protein levels after 24-hour treatment with DMSO or CC (10 μM). I. Effects of siRNA-mediated knockdown of ATF4 on LC3BII levels in stability of these NMD proteins. Several miRNAs including miR128, miR125, and miR433 have been shown to regulate NMD factors [6,47,48]; it will be interesting to determine in the future whether these miRNAs play a role in the CC-induced downregulation of NMD factors and attenuation of NMD efficiency.
At the cell level, both CC treatment and NMD inhibition induce autophagy (Fig 5)[30, 33]. The ability of CC to inhibit NMD suggests that NMD attenuation is part of the mechanism for the activation of autophagy by CC. Previously, it has been shown that autophagy induction after NMD inhibition is mediated in part by the stabilization of the mRNA of ATF4, a NMD target that promotes the transcription of the key autophagy factors LC3B and ATG5 [33,44]. However, we found that although CC treatment also induced and stabilized ATF4 transcripts, ATF4 was dispensable for autophagy induction by CC (Fig 5). Future work is needed to identify relevant NMD targets that promote autophagy in the presence of CC.
Our finding that CC inhibits NMD may have important implications for the use of CC as a chemical biology tool and as a potential therapeutic agent. The ability of CC to inhibit NMD may represent a mechanism for the many cellular phenotypes observed for CC. For example, in addition to autophagy, CC has been shown to induce apoptosis in many cancer cell lines and in vivo tumor models[28, [49][50][51][52]. Because NMD appears to be essential for human cells, its inhibition likely accounts partially for the effects of CC on cancer cell viability. CC has also been shown to induce cell differentiation, which was previously attributed in part to its inhibition of BMP type 1 receptors [53][54][55]. Because downregulation of NMD has been shown to promote multiple differentiation processes (e.g. embryonic stem cell differentiation, neurogenesis and myogenesis) by regulating the levels of many target mRNAs, including that involved in the BMP and TGF-β pathways in specific contexts [6,7,56,57], it is possible that NMD inhibition could contribute to CC-induced cell differentiation. On the other hand, the effects of CC on many other targets also limit its use as an inhibitor of NMD in research. Thus, caution needs to taken when interpreting results of CC treatment. In spite of its many off-target activities, CC or its derivatives with low toxicity may be effective in treating certain diseases by inhibiting NMD, as NMD inhibition may alleviate the symptoms of certain genetic disorders (e.g. β-thalassemia, cystic fibrosis, Hurler's syndrome, and Duchenne muscular dystrophy) that are caused by nonsense mutations in a single gene whose mutant protein products retain full or partial function [10]. Abrogation of NMD has also been suggested to induce antitumor immunity due to the expression of cancer antigens encoded by nonsense mRNAs [16,58]. Thus, CC or its derivatives may have the potential of improving immunotherapy for cancers that contain an increased level of nonsense mRNAs as a result of a high mutation load. Further investigation of the mechanism of action of CC in NMD inhibition will facilitate the development of CC or its derivatives as a therapeutic agent and aid our understanding of the physiological functions of NMD.

Key reagents
The key reagents used in this study, including plasmids, siRNAs, sgRNAs, antibodies, and chemicals are listed in Table 1. U2OS cells treated with DMSO or CC (10 μM) for 24 hours. J. ATF4 protein levels in WT or knockout U2OS cells. Because the basal level of ATF4 is below detection, cells were treated with 0.2 μM Thapsigargin (a known ER stressor that induces ATF4) for 4 hours before ATF4 western blot. K. Effects of ATF4 KO on LC3BII levels in WT or ATF4-KO cells treated with DMSO or CC (10 μM) for 24 hours. https://doi.org/10.1371/journal.pone.0204978.g005 Compound C inhibits NMD Table 1. Key reagents used in the study.

Reagent
Source Identifier
Lentiviruses expressing Cas9 or gRNAs used to knockout AMPK or ATF4 were generated by co-transfecting HEK293T cells with lentiviral vectors and packaging vectors (pCMV-VSVG and pPAX2) using TransIT-LT1 transfection reagent (Mirus). Cell culture medium containing lentiviruses was collected 48 and 72h after transfection, and used to infect target cells. Recombinant adenovirus expressing our NMD reporter was described before [19]. Target cells were infected with the reporter adenovirus for 24 hours before NMD analysis (see below).

NMD reporter assays
A dual color bioluminescence-based NMD reporter system described previously was used to measure NMD activity in human cells [19]. The reporter in the pBluescript (KS-) vector consists of a PTC-containing TCR minigene fused to CBR (CBR-TCRβ(PTC)) and a WT TCR minigene fused to CBG (CBG-TCRβ(WT)). NMD activity is measured by the ratios of CBR-TCRβ(PTC) to CBG-TCRβ(WT) at the protein and mRNA levels using bioluminescence imaging, western blot or RT-qPCR, as previously described [19,59]. U2OS cells stably expressing the NMD reporter system were described previously [19]. For analysis of NMD activity in Calu-6 and BJ cells, a recombinant adenovirus expressing the NMD reporter was used for infection. For bioluminescence imaging, cells were incubated with 150 μg/ml D-luciferin for 10 min at 37˚C, and bioluminescence signals were measured sequentially using IVIS 100 imager with appropriate open, red, green filters. Regions of interest (ROIs) were drawn over the images and bioluminescence signals were quantified using Living Image (Caliper) and Igor (Wavemetrics) analysis software packages as described previously [19,60]. Spectral unmixing was performed using an Image J plugin. For RNA analysis of CBR-TCRβ(PTC) and CBG-TCRβ(WT), total RNA was extracted using a Nucleospin RNA II Kit from Clontech (740955). Reverse transcription reaction was performed to synthesize cDNA using PrimeScript RT kit from Clontech (RR037A). RT-qPCR was performed using a two-step PCR protocol (melting temperature: 95˚C; annealing/extension temperature: 60˚C; cycle number: 40) on an ABI V117 real-time PCR system with PowerUp SYBR Green Master Mix (Thermo-Fisher). The mRNA levels of the housekeeping genes GAPDH were used for normalization. The sequences of all the primers used are listed in Table 2. Protein levels of CBR-TCRβ(PTC) and CBG-TCRβ(WT) (both of which are N-terminally HA-tagged) were measured by western blot with anti-HA antibodies using the Li-Cor Odyssey system. Cells were lysed in 50 mM Tris, 10% glycerol, 2% SDS, 5% β-mercaptoethanol. Protein lysates were run on SDS-page gels and transferred to a PVDF membrane, blocked with casein buffer and incubated with HA antibody (Biolegend, 901507). β-Actin detected with an antibody from Thermo-Fisher (MA5-15739) was used as a loading control.

Analysis of the expression and stability of endogenous NMD targets
Calu-6 cells expressing a mutant PTC-containing p53 mRNA were first treated with DMSO or CC for 24 hours. Subsequently, actinomycin D (5 or 10 μg/mL) was added to inhibit new mRNA synthesis. Total mRNA was isolated both immediately before and 6 hours after the addition of actinomycin D. mRNA levels of mutant p53 was measured using RT-qPCR. The percent of mRNA remaining (stability analysis) was determined as the percent of mRNA remaining after actinomycin D treatment, compared to that before actinomycin D treatment. https://doi.org/10.1371/journal.pone.0204978.t002 The stability of known physiological NMD targets including PISD, UPP1, FRS2, ATF4, PIM3, as well as transcripts of NMD factors UPF1, UPF2, SMG1, SMG5, SMG6, SMG7, and UPF3B were measured using RT-qPCR [19]. ORCL and HPRT, which are not NMD targets, were used as controls. Relative expression of different SC35 splice variants in U2OS cells was also measured using RT-qPCR. Sequences of the qPCR primers are listed in Table 2.

Knockdown and knockout of AMPKα or ATF4 in human cells
To deplete the catalytic subunits of AMPK (AMPKα1 and AMPKα2), U2OS cells were transfected with mixed siRNAs targeting AMPKα1 and AMPKα2, respectively, using the TransIT-siQUEST transfection reagent (Mirus). To knock out AMPKα1 and AMPKα2, a Cas9-expressing U2OS cell line was first generated by lentiviral transduction with pLenti-Cas9 Blast (Addgene 52962) followed by blasticidin selection for 5 days. sgRNAs in plentiGuide-Puro (Addgene 52963) targeting AMPKα1 and AMPKα2 were then introduced into cells for gene deletion by lentivirus transduction. Cells were then plated in 96-well dishes and individual knockout cell clones were screened and verified by western blot using AMPKα antibodies. Knockdown and knockout of ATF4 in U2OS cells were also performed by the siRNA transfection and CRISPR-Cas9 methods. Because basal expression levels of ATF4 are below detection in U2OS cells, knockdown or knockout was confirmed by western blot analysis after treating cells with CC or Thapsigargin, both of which induce ATF4 upregulation [61].

Immunoprecipitation and in vitro kinase assay
His-tagged, wild type (WT) or kinase dead (DA) SMG1 ectopically expressed 293T cells was immunoprecipitated using cobalt beads (HisPur™, ThermoFischer) in a buffer containing 300 mM NaCl, 50 mM Na 3 PO 4 (pH 8.0), 0.02% Tween, 0.25% NP40, and protease inhibitor cocktail (EDTA-free, Pierce). A recombinant GST-p53N fusion protein containing a N-terminal segment of p53 with the Ser15-phosphorylation site was expressed in E. coli and purified using FPLC with a GSTTrap column (GE Healthcare). In vitro kinase reaction was performed by incubating His-SMG1 (WT) or His-SMG1 (DA) with the GST-p53 substrate in the presence or absence of CC (10 μM) or caffeine (10 mM) at 30˚C for 2 hours in a kinase buffer (25 mM Tris HCl, pH 7.5, 10 mM β-Glycerophosphate, 0.2 mM Na 3 VO4, 10 mM MgCl 2 , 0.05 mM DTT). Reactions were terminated by the addition of SDS sample buffer. S15-phosphorylation of the GST-p53N substrate was detected by western blot using a phosphor-specific antibody (Cell Signaling Technology, 9284).

Immunofluorescence staining to detect LC3B foci
U2OS reporter cells were plated in 3.5 cm glass-bottomed dishes (MatTek corporation) and treated with DMSO or CC for 24 hours. Cells were then washed with phosphate buffered saline (PBS, pH 7.5), and then fixed using 4% paraformaldehyde (PFA) in PBS for 10 minutes. Subsequently, cells were permeabilized using 0.2% Triton X-100 in PBS followed by blocking for 1 hour with 10% normal goat serum in PBS. Cells were then incubated overnight with anti-LC3B antibodies (Cell Signaling Technology, 2775 at 1:200 dilution in PBS with 0.1% Triton X-100). After washing, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (Thermo-Fisher, A-11008 at 1:500 dilution) to detect LC3B foci. DNA was visualized with Hoechst 33342 staining (1 μg/ml). Fluorescence images were acquired using a Nikon Eclipse Ti-E inverted microscope with MetaMorph software, as described previously [62].