Fig. 2

(Upper panels) Time-course of DUX4c expression in primary FSHD muscle cells. FSHD primary muscle cells were grown and fixed with PAF either in proliferation (P) or after incubation in a differentiation medium at days 1 (D1), 3 (D3), or 6 (D6). DUX4c and Troponin T (TnT) were co-immunodetected using both rabbit and rat anti-DUX4c sera and the mouse anti-TnT antibody, followed by the appropriate secondary antibodies coupled to either Alexa Fluor 555 (red, rabbit anti-DUX4c), 647 (shown in purple, rat anti-DUX4c), or 488 (green, TnT). Arrows point to DUX4c cytoplasmic labeling and asterisks to nuclei which do not present DUX4c labeling. The strongest DUX4c nuclear staining is detected either in TnT-expressing cells (arrowheads) or in the nuclei of cells close to the ones expressing TnT ($). In proliferating cells (P), nuclei inside clusters are rounder and smaller (< 10 µm) compared to single cell nuclei of the same culture (also see Fig. S3) Circles highlight cytoplasmic accumulation of TnT that co-localizes with DUX4c either using the rat (D1) or the rabbit (D3 in Fig. S3) antiserum. Rectangles in D6 images indicate DUX4c detection using both DUX4c antisera in aligned nuclei of myotubes. These very close nuclei suggest that fusion has occurred recently [30]. The selected images correspond to magnification of rare regions boxed in Fig. S3A. Three to ten fields were analyzed per time in two cultures derived from two different muscles (Table S4)