Fig. 2: Inhibition of NMD system and differential RNA decay.

a Upper panel: schematic representation of the HSP110DE9-specific NMD reporter system used in this work. The NMD.reporter gene consisted of an in-frame HSP110 construction and contains the cDNA sequence from exon 1 to exon 8, intron 9, exon 10, intron 16, and exon 18. As in the case of the T₁₇ mutation of HSP110 located near the splice acceptor site of intron 8, a nonsense mutation appears in exon 10 due to the frameshift mutation caused by skipping of exon 9, making the exogenous mRNA a target of NMD. This construction is placed in an EBV stable vector. Lower panel: relative expression of HSP110DE9-PTC mRNA from NMD reporter stably transfected in the SW480 CRC cell line treated with several NMD inhibitors (siUPF1, cycloheximide (CHX), amlexanox, and NMDI-1). b Relative expression levels of TGFBR2, MSH3, or HSP110DE9 mRNAs determined by quantitative RT-PCR in CRC cell lines. After cycloheximide [CHX] treatment (4 h, 400 μg/ml). RNA expression levels compared to untreated cells [UT] in cell lines were analyzed with the TGFBR2 probe, [No Del] (SW480 and FET), Heterozygote [Htz] (HCT8 and RKO), Homozygote [Hmz] (HCT116 and LS174T); with the MSH3 probe, [No Del] (SW480 and HCT8), Heterozygote [Htz] = (LS174T), Homozygote [Hmz] (HCT116); with HSP110DE9 probes, [No Del] (SW480 and FET); [Del^S] (HCT116 and HCT8); [Del^L] (RKO and LS174T). Dashed line refers to the ratio calculated between treated and untreated cells with CHX. c Relative mRNA expression levels of MSI target genes (containing coding DNA repeats) as determined by quantitative RT-PCR in HCT116 (MSI) and SW480 (MSS) CRC cell lines transfected with siUPF1 (24 h post-transfection; top panel) or treated for 24 h with 5 µM amlexanox (lower panel). IGF2R is used as an internal control (not mutated in HCT116 and SW480 CRC cell line) for experimental conditions. All data are means ± SEM. Unpaired t-test was performed to determine significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001