Figure 5.

Effect of small molecules on GAA splicing. (A) Cells transfected with the MUT minigene were treated for 24 h with resveratrol (Resv), amiloride, tannic acid, kinetin or C6 pyridinium ceramide (C6). The relative expression of the N isoform was analyzed by real time PCR, using minigene-specific primers designed to amplify only this variant. The results are expressed as fold of N isoform detected in untreated cells. The abundance of the N isoform produced by cells transfected with the WT minigene is also shown for comparison. Data represent the means ± SD of three independent experiments. *P < 0.05. (B) Cultured fibroblasts from P1 and P2 patients were treated for 24 h with 200 µM of resveratrol and the relative abundance of the N variant was assessed by real time PCR. Results are expressed as percentage of the N variant detected in fibroblasts from a normal control. Data are means ± SD of three independent experiments. *P < 0.05. (C) Western blot analysis of the GAA mature protein (70 kD) in cultured fibroblast from a normal control (C) and the two P1 and P2 patients treated or not with resveratrol. (D) GAA enzymatic activity in cultured fibroblast from a normal control and P1 and P2 patients treated or not with resveratrol. Results are expressed as percentage of the activity detected in the normal control. Data represent the means ± SD of three independent experiments. *P < 0.05. (E) On the left, western blot analysis of endogenous SRSF proteins in HeLa cells treated or not with resveratrol. The positions of SRSF4, SRSF6 and SRSF1 are indicated. On the right, western blot analyses of DAZAP, YB-1 and U2AF65 factors. A western blot against tubulin was added to show loading control.