Figure 2
Cap-dependent translation is increased in OFD1-silenced cells. (a) OFD1-silenced cells (siOFD1; black bar) and control cells (Control; white bar) were transfected in triplicate with a plasmid overexpressing the Renilla luciferase under a constitutive promoter. Luciferase activity and Renilla mRNA levels were calculated and the ratio was reported in the graph. (b) On the top a scheme of the pRL-HCV-FL reporter plasmid is depicted. OFD1-silenced cells (siOFD1; black bar) and controls (Control; white bar) were transfected in triplicate with the reporter plasmid. The Renilla/Firefly luciferase light-unit ratio was calculated; the value for control cells was set at 1 and the fold change for all the samples was calculated and reported in the graph (left). The same experiment was performed in cells treated with rapamycin (RAPA, black bar) (graph on the right). The OFD1-silenced (siOFD1)/control (Control) cells ratio was calculated in untreated (NT, white bar) and treated (black bar) cells. (c) The accumulation of Renilla was rescued by overexpressing the murine form of Ofd1 (AAV.mOfd1), which is insensitive to siRNA. Data are presented as the mean ± SEM. Student’s t-test or wilcoxon test was used to calculate the p value as reported in Methods. **p-value < 0.02; ns: p-value > 0.05.
