Additional file 6: Figure S6. of Overexpression of the double homeodomain protein DUX4c interferes with myofibrillogenesis and induces clustering of myonuclei

DUX4c siRNA design and efficiency evaluation. A. Schematic representation of the DUX4c 3′UTR (positions from Genbank accession number AY500824). The STOP codon, two purine-rich regions which could be used as an alternative 3′end processing (as shown for some histone genes, [97]), the restriction sites EcoRI and AflIII (used for p3kb-DUX4c and 7.5-kb-DUX4c plasmid constructs, [26]) and the localization of the three designed siRNAs are indicated. Full-length DUX4c transcripts were already described in healthy and FSHD muscle cells [27]. B. Different DUX4c RNA ends found following transfection of C2C12 cells with p7.5-kb-DUX4c or in primary FSHD myoblasts (indicated by the asterisk). C. Evaluation of DUX4c knock-down using the three siRNAs shown in A. Human muscle TE671 cells were transfected or not (NT) with pCIneo-DUX4c expression vector (DUX4c) and with or without an siRNA targeting DUX4c as indicated (si1, si2, si3). Protein extracts were prepared 3 days later and analyzed by Western blot with the rabbit anti-DUX4c serum. Actin was used as a loading control. The panel with siRNA1 was previously shown in Ansseau et al. 2009 as part of Additional file 3: Figure S3 to confirm anti-DUX4c serum specificity. NT: not transfected. D, E. DUX4c expression in DUX4c-inducible stable TE671 cells [27] transfected with 20 nM DUX4c siRNA1. Four hours later, DUX4c expression was either induced or not with 1 μg doxycycline. C. Three or 5 days later, proteins were extracted, and 20 μg were separated on a 10% PAGE-SDS gel and transferred to a Western blot. DUX4c and actin were immunodetected. D. DUX4c detection by immunofluorescence (red) on parallel cultures. (PDF 90 kb)