Figure 3.

Ataluren induced translational read-through in patient-derived fibroblasts. (A) Fibroblasts of healthy donors, untreated USH2A patient-derived fibroblasts (untr.) and USH2A^G3142* patient-derived fibroblasts treated with Gentamicin (Gent, 1mg/ml) or Ataluren (Ata., 5 µg/µL, 10 µg/µL), respectively, were subjected to Western blot analysis. A stronger USH2A expression was detected in fibroblasts of healthy donors and patient-derived cells treated with 5 µg/µL Ataluren compared the untreated and Gentamycin-treated cells. (B) Quantification of Western blot analysis demonstrated the significantly increased USH2A protein levels in 5 µg/µL Ataluren treated USH2A patient-derived fibroblasts compared to untreated patient-derived cells (* p < 0.05). Three independent experiments are included. (C) Indirect immunofluorescence analysis of healthy fibroblasts and USH2A patient-derived fibroblasts. Immunofluorescence staining revealed a membranous USH2A localisation in fibroblasts of a healthy donor and in Ataluren-treated USH2A^G3142* patient-derived cells. A faint, punctuated USH2A protein expression was observed in untreated USH2A patient-derived cells. Treatment with Gentamicin partially restored USH2A localization at the membrane in patient-derived cells. Scale bar representative for the first three columns: 25 µm; scale bar representative for all zoom images: 5 µm. Three independent experiments were performed.