Figure 6: vtRNA1-1 modulates both the intrinsic and the extrinsic apoptosis pathway.

(a) qPCR identifies 10 apoptosis marker genes. Only mRNAs with expression levels that differ more than 2-fold between BL41 and BL41 cells expressing vtRNA1-1 (after a 4.5 h staurosporine treatment) were taken into consideration. Upregulation is reflected by a positive and downregulation by a negative fold-change value. The data shown derive from averaging two biological replicates. (b) Protein levels of Bcl-xL, ARC and the cleaved caspases Casp-9 and Casp-3 in BL41 cells expressing vtRNA1-1 or vtRNA1-2 in the absence (−) or presence (+) of staurosporine (Stau) were assessed by western blot analyses. Cleavage of Casp-8 was monitored after Fas-L treatment. Proteins GAPDH or L9 served as loading controls. See also Supplementary Fig. 10. (c) Western blot analyses (upper panel) were used to monitor the kinetics and levels of phosphorylated IκB expression in BL41 cells, or in BL41 cells expressing vtRNA1-1 or vtRNA1-2, respectively, as a function of TNFα incubation (in minutes). Hsp90 served as loading control. The same cell lines were used to test for Bcl-xL mRNA expression by qPCR (lower panel). The data represent the mean and standard deviation of two independent experiments. Locations of molecular weight markers (kDa) are shown on the left (b) or right (c), respectively. (d) A putative model showing how vtRNA1-1 confers apoptosis resistance by modulating the intrinsic as well as the extrinsic pathways. When vtRNA1-1 levels are high (green arrow), such as in the presence of EBV infection and LMP1 signalling (orange), ARC and Bcl-xL become upregulated (green arrows). These proteins subsequently contribute to inhibiting the intrinsic as well as the extrinsic apoptotic pathway resulting in reduced levels of cleaved caspases 9 (intrinsic), 8 (extrinsic) and 3 (both pathways). In the latter case Bcl-xL neutralizes caspase-8 processed Bid at the outer mitochondrial membrane that links the extrinsic pathway to the mitochondrial pathway required for full effector caspase activation thus leading to resistance to Fas Ligand-induced cell death.