Figure 6.

Allele-specific silencing of the exon-16 skipped mRNA improves the quality of the newly formed collagen VI matrix. (a) Immunofluorescence staining of collagen VI (green), co-stained with fibronectin (red) as secreted and deposited in scrambled small interfering RNA (siRNA)-treated samples (one patient, UCMD1, and unaffected cell line), or in siRNA-1 and siRNA-2–treated samples (UCMD1). Four consecutive siRNA treatments (once every 48 hours) 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 allowed 3 days for collagen VI to be secreted and deposited into the matrix. Images were taken on a confocal microscopy system. Scale bar = 12 μm. (b) Insets from images in a. (c,d) Qualitative assessment of collagen VI microfibrils. Five independent observers blinded to the treatment condition scored 85 different collagen VI microfibril images, selected from four different conditions (UCMD1 cells treated with siRNA-1, siRNA-2, or control siRNA-Scr, and cells from an unaffected individual treated with control siRNA-Scr). The aspect of the microfibrils was categorized as either “discontinuous/speckled”, “partially continuous”, or “continuous/solid”. (c) Examples of collagen VI microfibril images that were unambiguously scored as “discontinuous/speckled”, “partially continuous”, or “continuous/solid”. (d) Histogram of combined scoring results depicting the fraction of images that were scored as either “discontinuous/speckled”, “partially continuous”, or “continuous/solid” for each treatment condition.