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. 2015 Sep 18;23(3):469–483. doi: 10.1038/cdd.2015.114

Figure 4.

Figure 4

Identification of PML 5′-UTR (-100->-1) as an IRES activated by the TNFα–MNK1 axis. (a) A schematic representation of bi-cistronic pRF plasmids used in the transient transfection reporter assays. (b) The effects of PML 5′-UTR (-100->-1) on reporter activity. The relative ratio of Firefly luciferase/Renilla luciferase intensity is plotted. The results are mean±S.D. in triplicates (n=3). (c) Determination of possible alternative splicing of the transcripts of PML 5′-UTR (-100->-1)-pRF. The cDNAs prepared from pRF or PML 5′-UTR (-100->-1)-pRF transfected HUVECs subjected to PCR using primer pairs as indicated in (a). The final PCR products were separated on a 0.8% agarose gel, stained by EtBr and images recorded. (d) Knockdown of RLuc in bicistronic system reduces FLuc driven by EMCV IRES and PML 5′-UTR (-100->-1) but not by PML 5′-UTR (-140->-1). (e) The reporter activity of PML 5′-UTR (-100->-1) is not derived from ribosomal read-through. HeLa cells were transiently transfected with engineered pRF and β-gal plasmids as indicated. At 48 h post transfection, the Firefly luciferase and Renilla luciferase activity were measured and normalized to the transfection control, β-gal. The luciferase activity of PML 5′-UTR (-100->-1)-pRF was set as 1. The results are mean ±S.D. in triplicates. (f) EMCV IRES and PML 5′-UTR (-100->-1) harbors IRES activity as evidenced by transfection of in vitro transcribed mRNAs into HeLa cells. The relative activity of RLuc and FLuc was normalized to the corresponding mRNA levels 9 h post transfection. (g) TNFα enhances PML 5′-UTR (-100->-1) activity in a MNK1-dependent manner. shCtrl and shMNK1 knockdown HeLa cells were transiently transfected with PML 5′-UTR (-100->-1)-pRF plasmid for 72 h, treated with TNFα at the indicated times and cells harvested. The relative activity of Firefly luciferase over Renilla luciferase was plotted. The results are mean ±S.D. in triplicates (n=3). (h and i) Ectopic expression of eIF4E inhibitor, 4E-BP or a mutant defective in phosphorylation by mTORC1, 4E-BP (5A), does not compromise TNFα-mediated PML protein accumulation or PML IRES activation. HeLa cells were transiently transfected with PML 5′-UTR (-100->-1)-pRF plasmid with the indicated plasmids for 48 h, treated with TNFα for 20 h and cells harvested. (h) Endogenous PML protein in pEBG, pEBG-4E-BP and pEBG-4E-BP (5A) transfected cells were determined by western blottings. The relative PML protein expression was normalized by β-actin levels and (PML)/(β-actin) in the absence of TNFα was set as 1. The relative fold induction of PML protein abundance by TNFα is shown in each sample. Both long (L.E.) and short (S.E.) exposures of PML western blot are shown. (i) The relative effect of TNFα on (FLuc)/(RLuc) of PML 5′-UTR (-100->-1) reporter in transfected samples is plotted. The results are shown as mean±S.D. (n=4)