Figure 4.

Lentiviral delivery of ExSpeU1 rescues ELP1 splicing and protein expression in FD patient fibroblasts. (A) Endpoint PCR analysis of the ELP1 splicing pattern in FD fibroblasts treated with lentiviral particles expressing the ExSpeU1 Ik10 (Lentivirus U1 Ik10) and the wild-type U1 snRNA (Lentivirus U1 wt) at different multiplicities of infection. The upper band of 202 bp corresponds to transcripts including the exon 20, the lower band of 128 bp corresponds to exon 20 skipping. The percentage of exon 20 inclusion of three independent experiments is reported as mean±SD. (B) Quantitative analysis of ELP1 mRNAs’ isoforms by qPCR. The histograms represent the fold change of total (Tot ELP1), full-length (ELP1 FL) and exon 20 skipping (ELP1 Δ20) ELP1 mRNAs compared to the untreated cells (n.t.). A schematic representation of each amplicon is shown. Statistical analysis was performed using a two-ways ANOVA (***P<0.0001, **P < 0.001; ns, not significant). (C) ELP1 protein detection by western blot with human-specific primary antibody in FD (Fibro FD) and normal (Fibro WT) fibroblasts. α-tubulin was used for internal normalization and ELP1 fold change of FD-treated fibroblasts was referred to normal fibroblasts.