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Additional file 5: Figure S4. DUX4c immunofluorescence intensity variation during primary myoblast differentiation time-course.

Additional file 15: Figure S14. Negative PLA controls in healthy and FSHD myofibers.

Additional file 3: Figure S2. Validation of the rat anti-DUX4c serum.

Additional file 7: Figure S6. DUX4c and laminin-α2 detection by co-immunofluorescence in FSHD muscle sections.

Additional file 2: Figure S1. DUX4C mRNAs and protein in muscle cells.

Additional file 8: Figure S7. DUX4c-desmin co-detection by immunofluorescence in FSHD muscles.

Additional file 9: Figure S8. DUX4c is immunodetected in regenerating myofibers.

Additional file 6: Figure S5. DUX4c detection by immunohistochemistry in muscle sections and histological alterations in FSHD muscles.

Additional file 4: Figure S3. Time-course of DUX4c expression in primary FSHD muscle cells.

DUX4c inhibition in myoblasts decreases mitosis. DUX4c inhibition causes cell proliferation defects. FSHD and control primary myoblasts were transfected with siRNAs targeting either the DUX4 or DUX4c mRNA or a non-targeted (nt) siRNA. Top: microscope pictures in white light taken of representative fields of each culture. Bottom: The number of mitosis present per field was counted in each culture (10 fields per culture). Histograms show the mean of the mitosis number present per field in each culture. ***p

Cytoskeleton-associated protein quantification in healthy and FSHD myotubes : reorganization of the proteomic data presented separately in Tassin et al (2012: Figure 3, supplemental tables; and unpublished data). Data were subdivided in the three classes of cytoskeletal proteins : actin thin filaments, intermediate filaments and microtubules. In summary, primary healthy, aFSHD (atrophic) and dFSHD (disorganized) myotubes were harvested 4 days after induction of differentiation and protein extracts (TE: total extract; NE: nuclear extract) were analysed by post-digest ICPL coupled to LC-MS/MS. UniProt accession number; Hugo Gene symbol; Protein name; H/L: fold change (v: identified protein without quantification); SD: geometric standard deviation (n.d.: not determined when an abnormal distribution is observed); #: number of peptides used for quantification; *: statistical significance (p

dFSHD myotubes present abnormal clusters of nuclei overexpressing DUX4c, cytoplasmic DUX4c, and α-tubulin as well as α-crystallin B chain delocalization. A. Healthy and FSHD primary myoblasts were differentiated and fixed 6 days later, and DUX4c (red) and α-tubulin (green) were detected by immunofluorescence. Clusters of nuclei are surrounded by abnormal or almost an absence of α-tubulin. B. The α-crystallin B chain is highly expressed in the cytoplasm of dFSHD myotubes, in contrast to the low nuclear expression observed in healthy myotubes. (PDF 266 kb)

DUX4 myogenic enhancer (DME1 and 2) might interact with the DUX4c gene. The DUX4 forward primer used in a 3C capture experiment [79] is highlighted in a common region in DUX4 and DUX4c ORF sharing 100% identity (Accession numbers AF AF117653 and AY500824). In Himeda et al. [79], RT-qPCR was performed using this primer in combination with either DIR1 (containing DME1) or DIR2 (containing DME2) primer to confirm DUX4-DME1 or DUX4-DME2 interactions. The BlgII site (boxed) used to digest chromatin-linked DNA regions is also shown and is located 226-bp downstream of the 5’end of the DUX4 primer. The region between DUX4 primer and the BglII site share 100% identity. (PDF 287 kb)

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)

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)

DUX4c overexpression induces β-catenin. Healthy primary myoblasts were transfected with pAC1M2-DUX4c and differentiated 48 h later. After the first myotubes formed (day 3), DUX4c expression was induced by the addition of 100 ng or 1 μg of doxycycline to the culture medium in parallel to the cultures showed in Fig. 2. At day 8, myotubes were fixed and proteins were extracted, separated, transferred to a Western blot, and the indicated proteins were immunodetected as described in Fig. 1b. Histograms: densitometry of the immunoreactive bands normalized to the actin levels (Ponceau red) in each sample. Dox: doxycycline. (PDF 115 kb)

siRNA1 specifically silences endogenous DUX4c. RNA interference as described in Fig. 6. Endogenous DUX4c was detected by immunofluorescence (red). (PDF 51 kb)

DUX4c overexpression induces the formation of ring-like clusters of nuclei and detection of DUX4c in nuclei and the cytoplasm. Healthy primary myoblasts were transfected with pAC1M2-DUX4c and differentiated 48 h later. After formation of the first myotubes (day 3), DUX4c expression was induced by the addition of doxycycline (0 ng, 100 ng, or 1 μg) to the culture medium. At day 8, myotubes were fixed. DUX4c (red) and troponin T (green) were detected by immunofluorescence. Nuclei were labeled with DAPI. The circle indicates the cytoplasmic detection of DUX4c at one myotube tip, where troponin T expression was highest in the elongated myotubes. Scale bar: 30 μm. (PDF 89 kb)

The study aims 1) to examine whether items of the brief geriatric assessment (BGA) or their combinations predicted the risk of unplanned emergency department readmission after an acute care hospital discharge among geriatric inpatients, and 2) to determine whether BGA could be used as a prognostic tool for unplanned emergency department readmission.A total of 312 older patients (mean age, 84.6 ± 5.4 years; 64.1% female) hospitalized in acute care wards after an emergency department visit were recruited in this observational prospective cohort study and separated into 2 groups based on the occurrence or not of an unplanned emergency department readmission during a 12-month follow-up period after their hospital discharge. A 6-item BGA was performed at emergency department admission before the discharge to acute care wards. Information on incident unplanned emergency department readmission was prospectively collected by phone call and by consulting the hospital registry. Several combinations of items of BGA identifying three levels of risk of unplanned emergency department readmission (i.e., low risk, intermediate risk and high risk) were examined.The unplanned emergency department readmission was more frequently associated with a temporal disorientation (P=0.004). Area under receiver operating characteristic curves of unplanned emergency department readmission based on BGA items and their combinations ranged from 0.53 to 0.61. The best predictor of unplanned emergency department readmission was the temporal disorientation (hazard ratio1.65, P0.035), which defined the high-risk group. Inpatients classified in high-risk group of unplanned emergency department readmission were more frequently readmitted to emergency department than those in intermediate- and low-risk groups (P log Rank0.004). Prognostic values for unplanned emergency department readmission of items and their combinations were poor with sensitivity below 67%, specificity ranging from 36.4 to 53.7, and positive likelihood ratio below 1.4.The items of BGA and their combinations were significant risk factors for unplanned emergency department readmission, but their prognostic value was poor.