Additional file 5: Figure S5. of Overexpression of the double homeodomain protein DUX4c interferes with myofibrillogenesis and induces clustering of myonuclei

DUX4c overexpression induces expression of FSHD markers. FSHD and healthy primary myoblasts were transfected with the indicated pCIneo expression vectors. A. Total protein extracts were prepared 48 h after transfection. A 30 μg sample of each extract were separated by electrophoresis, transferred to a Western blot, and the indicated proteins immunodetected. This image was used in Vanderplanck et al. (2011) for FSHD, healthy, and DUX4-overexpressing myoblasts. We only added the lane corresponding to DUX4c-overexpressing myoblasts. B. Total protein extracts were prepared 48 h after transfection (top) or 8 days after the induction of differentiation (middle). A 30 μg sample of the extracts was separated via electrophoresis, transferred to a Western blot, and immunodetected. Actin was stained with Ponceau red on the same membrane before immunodetection and was used as the loading control. N.B.: In these conditions, neither endogenous DUX4 nor DUX4c could be immunodetected with MAb 9A12. C. Immunodetection of MuRF1 in healthy FSHD and in DUX4- or DUX4c-overexpressing primary myotubes fixed 7 days after the induction of differentiation. Scale bar: 20 μm. D. γ-Catenin (JUP) mRNA quantification by RT-qPCR in RNA of healthy and FSHD primary muscle cells. The quantity of γ-Catenin mRNA was expressed relative to its amount in healthy cells and set to 1. The means and standard errors are indicated. (PDF 452 kb)