Fig. 7

SC35 speckles were disrupted in many nuclei by expression of DUX4-FL from its endogenous promoter. a–d” Endogenous expression of DUX4-FL (green) in FSHD myotubes caused SC35 speckles (red) to show an altered morphology. Arrows indicate nuclei that expressed DUX4-FL, and asterisks indicate nearby nuclei that were DUX4-FL-negative. In panel d, the dotted line indicates where empty space was cropped from the image so that two nearby neighboring nuclei could be juxtaposed for presentation. The most common changes to SC35 speckles in the DUX4-FL-positive nuclei were the appearance of larger aggregates and/or more intense staining. Less common changes included loss of most speckles (e.g., rightmost nucleus in row a) and disorganized speckles (e.g., row b). SC35 speckles were disrupted in both nuclei with punctate DUX4-FL staining (rows a, c) and in nuclei with uniform DUX4-FL staining (row b). In nuclei with punctate DUX4-FL (rows a and c), there was little or no overlap between DUX4-FL and SC35 staining. Some nuclei also showed little effect of DUX4-FL (e.g., leftmost nucleus in row a). e Quantitation of SC35 speckle morphology. As described in the text, a blind test was used to classify speckle patterns into three groups: (i) similar to the majority of controls (normal, light gray bars), (ii) maybe different from controls (medium gray bars), or (iii) obviously different from controls (dark gray bars). Nuclei that expressed either endogenous or exogenous DUX4-FL had much higher frequencies of obviously different SC35 speckle patterns, compared to nuclei in healthy control cultures or to nuclei that expressed exogenous DUX4-S or PITX1. Bar in a = 20 μm