Figure 1

Microscopy cell-based assay to screen for SMN protein in nuclear-body Cajal bodies (CBs) of SMA patient-derived fibroblasts. (A) Immunofluorescent staining of SMN and CB-marker coilin protein in flunarizine treated (2 μg/ml) and untreated (DMSO) immortalized Type I SMA fibroblasts using rabbit polyclonal anti-SMN and mouse monoclonal anti-coilin antibodies. (B) Analysis of the immunolocalization of SMN and snRNPs (TMG-capped snRNAs) into CBs of immortalized Type I SMA fibroblasts for seven molecules. (C) Total protein extracts from immortalized type I SMA fibroblasts treated with 2 μg/ml of each molecule were separated by SDS-PAGE and SMN protein was detected by western blotting using tubulin as loading control. Odd and even numbers correspond to 5 and 15 μg of proteins, respectively. (D) The ability of the molecule to recruit SMN to CBs in immortalized SMA fibroblasts was tested in the presence of decreasing concentrations of each molecule, the 1:1 dilution being 2 μg/ml. (E) Analysis of the immunostaining of SMN in CBs of drug-treated primary fibroblast cultures from patients affected with the three forms of SMA disease. See Supplemental Table 3 for statistical analyses. (F) HeLa cancer cells were treated with 4 (2 μg/ml) and 20 μM flunarizine for 4 h and compared to 1 or 5 μl/ml DMSO. An increase in the proportion of cells with 3 or 4 CBs per nucleus is observed with both concentrations of flunarizine. Data represent the mean values ± SEM. Chi2 test, ***p < 0.001. Scale bar, 10 μm.