Figure 5.

The Efficiency of ASOs on 2′-MOE Backbone in Restoring ECM Collagen VI Protein

(A) Representative images of immunofluorescence staining of collagen VI protein (in green) and nuclei (in blue) in patient skin fibroblasts treated with 2′-MOE ASOs. Pictures were captured under fluorescence microscopy at 10× (top panel) and 40× (lower panel) original magnification. Scale bars: 100 μm for 10× magnification and 50 μm for 40× original magnification. (B) The structure of ECM collagen VI expression in fibroblasts from healthy control and UCMD patients treated with 20 nM ASOs. Images were captured under confocal microscope. In healthy condition, collagen VI forms the liner microfibrils (white arrow) in ECM, while in UCMD patients the linear structure is replaced with discontinuous and speckled microfibrils (yellow asterisk). Treatment of ASO-5, ASO-6, ASO-5/6, or ASO-9 restored the collagen VI deposition pattern to linear microfibrils. Scale bar: 10 μm. (C) Mean intensity and the area covered by collagen VI were quantified in fibroblasts treated with a single transfection of ASO-scr, ASO-5, ASO-6, ASO-5/6, and ASO-9 at 20 nM. Data represent mean ± SD. Data were analyzed by one-way ANOVA followed by post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). (D) The beaded microfibril network of collagen VI protein was visualized by scanning electron microscopy in UCMD fibroblasts treated with 20 nM ASO-5 or ASO-5/6. Scale bar: 100 nm.