Figure 4.

NCL targeting by N6L increases 5′TOP and 5′TISU mRNA translation by activating mTOR pathways. (A)—Translation Index (T.I) for 5′TOP-containing mRNA under N6L treatment (A, right). (B)—Venn diagram comparing 5′TOP-gene overlap between our TI UP mRNAs and 5′TOP gene lists from Gentilella et al. 2017 [33]. (C)—Summary table showing the distribution of 5′TOP and non-5′TOP mRNAs according to their transcriptional expression (up, down or stable). (D)—mPDAC cells were treated (T) or not (NT) with N6L for 48 h, following which RPL36 and RPS3 expression at the protein level was analyzed using Western blotting (D, upper panel) and quantified in relation to H3 (D, lower panel). (E)—Cartoon showing the design of 5′TOP firefly-luciferase reporter constructs, with the first C of the TOP motif as the +1 transcription start site (TSS). (F)—Fold change of firefly-luciferase activities under 5′TOP motifs of Rpl36 and Rps3 mRNAs upon 48 h of increasing concentrations of N6L treatment in mPDAC, after normalization to mPDAC NT. * p < 0.05; ** p < 0.01 and n.s. for not statistically significant, as determined by Student t-test on the final data point.