Figure 4

Effects of flunarizine on the expression and splicing profiles of candidate genes in SMA mice. (A) The snRNP-specific reduction of snRNAs in brain and spinal cord of SMA mice is modulated by flunarizine. The snRNA levels are determined by RT-qPCR and the relative amount is presented as percent of the vehicle-treated controls. 5 S and 5.8 S were used as controls for normalization as described²². (B) RT-qPCR analysis of different genes shows that flunarizine reduces the retention of SMN2 intron3 in brain of SMA mice. Four genes were used as controls for normalization in brain (RPL13A, PPIA, HPRT1, SDHA) and spinal cord (RPL13A, PPIA, HPRT1, ACTB). (C–F) The expression ratio between exons or isoforms of SMN2, TTYH3, AGRN and SNAP25 genes. SMN2 and TTYH3 were analysed by RT-qPCR whereas AGRN and SNAP25 were by RT-PCR experiments. No significant differences of SMN2 exon 7/ex 4 ratios were found between flunarizine- and vehicle -treated SMA mice in panel 4 C (p = 0.007). Total RNA was prepared from tissues of flunarizine (Flz)- and vehicle (V)-treated controls and SMA mice at P11. Three mice per group. Data represent the mean values ± SD (errors bars). Statistical test used is the two-way Anova followed by the Tukey’s multiple comparisons test (GraphPad).