Tobramycin is a suppressor of premature termination codons Nicola Altamura, Rosa Castaldo, Alessia Finotti, Giulia Breveglieri, Francesca Salvatori, Cristina Zuccato, Roberto Gambari, Giulia Chiara Panin, Monica Borgatti  Journal of Cystic Fibrosis  Volume 12, Issue 6, Pages 806-811 (December 2013) DOI: 10.1016/j.jcf.2013.02.007 Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Read-through properties of Tobramycin, Gentamicin and G418 at a premature termination codon relative to NMD+ untreated yeasts (0 NMD+). A dual-luciferase reporter plasmid expressing the Renilla and Firefly luciferase open reading frames, separated by the UAG stop codon or the CAG sense codon [11] was introduced into yeast strains with either functional (NMD+, left side) or abolished NMD (NMD−, right side). Transformants were growth, either in the absence or in the presence of the indicated concentrations of the aminoglycosides A) Tobramycin, B) Gentamicin or C) G418. Yeast lysates were prepared for a dual-luciferase reporter assay. Read-through percentages were calculated as the nonsense Firefly/Renilla activity ratio normalized to the sense Firefly/Renilla activity ratio×100 and reported relative to control (untreated yeasts, NMD+) set arbitrarily to 1. Mean±SD values were determined for each percentage activity for at least four independent experiments. *p<0.05 by Student t test. D. Analysis of the firefly luciferase (left side) and CAN1-100 (right side) mRNA levels in yeast by real time PCR. Yeasts, with active (NMD+) or abolished NMD (NMD−), carry firefly luciferase gene with UAG nonsense codon (black boxes) or CAG sense codon (white boxes) and CAN1-100 (CAN1 gene with nonsense codon). Data represent the average of three independent experiments±SD. Journal of Cystic Fibrosis 2013 12, 806-811DOI: (10.1016/j.jcf.2013.02.007) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Read-through relative to NMD+ untreated yeasts or NMD− untreated yeasts of Tobramycin, Gentamicin and G418 at nonsense codons in yeast strains with either functional or abolished NMD. Yeast strains with functional (NMD+, on the left) or abolished NMD (NMD−, on the right) were transformed with a dual-luciferase reporter plasmid in which the two luciferases were separated by A) UAG stop codon or CAG sense codon; B) UGA stop codon or CGA sense codon; and C) UAA stop codon or CAA sense codon. Read-through percentages, calculated as the nonsense Firefly/Renilla activity ratio normalized to the sense Firefly/Renilla activities ratio×100, are reported relative to NMD+ untreated yeasts (0 NMD+) or NMD− untreated yeasts (0 NMD−). Mean±SD values were determined for each percentage activity for at least four independent experiments. *p<0.05 by Student t test. Journal of Cystic Fibrosis 2013 12, 806-811DOI: (10.1016/j.jcf.2013.02.007) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions