BART6-3p-regulated pathways in BL-derived cell lines. (a) The expression of BART6-3p was evaluated in two EBV-negative BL-derived cell lines (Akata 2A8 and Ramos) and two EBV-positive BL-derived cell lines (Akata and Raji), to select that expressing BART6-3p at the highest level. The Akata cell line shows higher expression level of BART6-3p. (b): qRT-PCR of BART6-3p target genes following BART6-3p inhibition. A BART6-3p antagomir was transfected in Akata cells and the expression level of p80, gp130 and PTEN was evaluated 24 hrs post-transfection. Cells transfected with BART6-3p inhibitor showed a marked up-regulation of p80, gp130 and PTEN, in respect with cells transfected with the negative control (NC), confirming regulation of these genes by BART6-3p. As a control, down-regulation of the endogenous BART6-3p was observed following transfection with the antagomir. The graph is representative of three different qRT-PCR experiments. Error bars represent standard deviation between duplicates. (c): Inhibition of BART6-3p by its antagomir results in the activation of the downstream pathways of the IL-6 receptor (p80 and gp130) and PTEN. The expression of p80, gp130 and PTEN was evaluated at the protein level by WB. In addition, two downstream signaling pathways were also evaluated following BART6-3p inhibition, the activation of NF-κB, which occurs through IκBα phosphorylation, and the decrease of the phosphorylated form of Akt, which acts downstream from PTEN. Protein levels were evaluated 48 hours post-transfection by Western blotting. The bands were then analyzed by the Image J software and relative expression levels were normalized to those of β-actin. Activation of NF-κB is consequent to IκB-α phosphorylation; PTEN activity results in the negative regulation of Akt, documented by a lower phosphorylation level. EBNA1 expression was measured as a control for EBV-positivity. The figure is representative of three different experiments.