Figure 5.

Allele-specific silencing of the exon 16-skipped mRNA increases extracellular collagen VI matrix deposition in cultured congenital muscular dystrophy type Ullrich (UCMD) fibroblasts. (a) Representative images of immunofluorescence staining of collagen VI matrix, as secreted and deposited by one normal (Unaffected) and two UCMD fibroblast cell lines (UCMD1 and UCMD3). Four consecutive small interfering RNA (siRNA) treatments (once every 48h) were performed with either siRNA-1, siRNA-2, or the scrambled control siRNA-Scr. Following the last treatment, culture medium was supplemented with L-ascorbic acid, and cells were maintained in culture 3 to 5 days to allow for collagen VI secretion and matrix deposition. Stacks of confocal microscopy images were acquired (section thickness of 0.5 μm, one image every 0.5 μm), and are presented as a merge. Scale bar = 38 μm. (b) Total collagen VI volume in the matrix was quantified in each image (between 6 and 10 images per experiment, where each experiment was performed either once or in duplicate) by measuring the sum of the volume of all objects displaying a longest axis greater than 15 μm, using Volocity. Total volume was normalized by the number of nuclei in each image, and plotted as dot-plot. Bars indicate the median and interquartile range. Kruskall–Wallis and Dunn's tests were applied. *P < 0.05.