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Fig. 7 | Skeletal Muscle

Fig. 7

From: The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins

Fig. 7

DUX4c interacts with C1qBP in FSHD myofibers. DUX4c and C1QBP interaction was determined by in situ proximity Ligation Assay (Duolink PLA) performed on fixed FSHD muscle sections (healthy control sections and negative controls are presented in Fig. S13A, B, C), using the rabbit anti-DUX4c serum and a mouse anti-C1QBP serum, followed by appropriate secondary antibodies coupled either to a plus- or a minus-DNA probe. If at 40-nm maximal distance both probes ligate, PLA signal can be amplified and detected by hybridization with a fluorochrome-coupled oligonucleotide, which corresponds to red dots. Laminin-α2 and the F-actin-binding phalloidin (to highlight the sarcoplasm) are detected with specific antisera, followed by secondary antisera coupled either with Alexa-488 (green, laminin-α2) or -647 (far red, F-actin), respectively. Staining was observed by confocal microscopy. (Upper panel) 3D reconstruction of a muscle section area with one myofiber containing a central nucleus (#). Boxes represent clear PLA signals with large dots in clusters and circles indicate the signals we have arbitrary set as nonspecific: dots not in a cluster on several Z axes (see example in box 1). (Bottom panels) Different focal depths or magnifications (17 images with steps of 0.25 μm in the Z axis). Box 1 corresponds to two distinct depths focusing on two specific PLA signals (arrowheads) either near a cluster of nuclei at a myofiber tip or at a single nucleus periphery. Both are in areas without phalloidin detection suggesting these nuclei belong to cells with a very small cytoplasm, as laminin-α2 staining is detected either surrounding the clustered nuclei or as a line inside a myofiber next the single nucleus, or is co-detected with the PLA signal (stars). The yellow arrowhead points to a PLA signal that presents a shape distinct from the surrounding laminin-α2 and might be localized at the tip of a satellite cell. Box 2 focusses on a region (two magnifications at two depths) where many PLA signals are found on both sides of an elongated nucleus with co-detection of intense laminin-α2 dots. This cell partially surrounds a myofiber and could be an activated satellite cell. Arrows point to distinct PLA signals and laminin-α2 dots. PLA was performed with the anti-DUX4c and-C1qBP primary antisera pairs on muscle sections from 4 patients with FSHD (in parallel to muscle sections from 3 healthy controls, see Fig. S13C)

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