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(Figure S1). To be sure to clone an shRNA really efficient in suppressing UPF1, we first tested three different siRNAs, in a transient way using siPORT™ NeoFX™ Transfection Agent (Thermo Fisher Scientific, Waltham, MA, USA). After 2 days incubation, we evaluated the expression of UPF1 by Real Time qPCR assay. The results show the UPF1 fold expression in presence of each siRNA compared to control cells transfected with a scramble siRNA. The UPF1c showed the best suppression effect (Figure S1). (Figure S2). The UPF1- clone 3 derived from β039 globin K562 cells (β039.m5) has been tested with different aminoglycosides (geneticin, tobramycin and gentamicin), confirming its application as cellular model for screening of read-through molecules. As far as tobramycin, it was able to induce read-through activity with a complete NMD suppression in yeast models [24, 25]. On the other hand, the read-through activity of gentamicin is firmly established [6, 19]. For these reasons, we treated the cells with 400 ng/μl geneticin (G418), 1400 ng/μl gentamicin or 1000 ng/μl tobramycin for 72 h [6, 24]. Then the cells were labeled with an antibody against β globin, followed by FACS analysis (see Methods section). Figure S2 shows the averages (± SD) of the data obtained in three independent experiments, representing the percentage of fluorescent cells (producing β globin chains) and the relative increase in the treated samples compared to negative controls. With G418, the β039.m5 control cells show a 5.4% increase in the proportion of fluorescent, which was 10.9% in the derived UPF1- clone 3 (Figure S2A and Fig. 6a). After gentamicin and tobramycin treatment, the β039.m5 control cells displayed an increase of 3.3 and 1.2% in the proportion of fluorescent cells, respectively. While in the UPF1- clone 3, the increase was 8.9 and 4.7% after gentamycin or tobramycin treatment (Figures S2B-C). (DOCX 635 kb)

(Figure S1). To be sure to clone an shRNA really efficient in suppressing UPF1, we first tested three different siRNAs, in a transient way using siPORT™ NeoFX™ Transfection Agent (Thermo Fisher Scientific, Waltham, MA, USA). After 2 days incubation, we evaluated the expression of UPF1 by Real Time qPCR assay. The results show the UPF1 fold expression in presence of each siRNA compared to control cells transfected with a scramble siRNA. The UPF1c showed the best suppression effect (Figure S1). (Figure S2). The UPF1- clone 3 derived from β039 globin K562 cells (β039.m5) has been tested with different aminoglycosides (geneticin, tobramycin and gentamicin), confirming its application as cellular model for screening of read-through molecules. As far as tobramycin, it was able to induce read-through activity with a complete NMD suppression in yeast models [24, 25]. On the other hand, the read-through activity of gentamicin is firmly established [6, 19]. For these reasons, we treated the cells with 400 ng/μl geneticin (G418), 1400 ng/μl gentamicin or 1000 ng/μl tobramycin for 72 h [6, 24]. Then the cells were labeled with an antibody against β globin, followed by FACS analysis (see Methods section). Figure S2 shows the averages (± SD) of the data obtained in three independent experiments, representing the percentage of fluorescent cells (producing β globin chains) and the relative increase in the treated samples compared to negative controls. With G418, the β039.m5 control cells show a 5.4% increase in the proportion of fluorescent, which was 10.9% in the derived UPF1- clone 3 (Figure S2A and Fig. 6a). After gentamicin and tobramycin treatment, the β039.m5 control cells displayed an increase of 3.3 and 1.2% in the proportion of fluorescent cells, respectively. While in the UPF1- clone 3, the increase was 8.9 and 4.7% after gentamycin or tobramycin treatment (Figures S2B-C). (DOCX 635 kb)