mDUX and myogenic regulators.

<p>qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using 500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and Pax7 when mDUX is induced with various concentrations of doxycycline.</p

Toxicity of mDUX.

<p>(A) Morphology of iC2C12-mDUX cells induced for 24 hours (Dox) with 500 ng/ml doxycycline. The majority of induced cells were detached and floating after 24 hours. (B) ATP assay for analysis of viability in iC2C12-mDUX cells induced with various concentrations of doxycyline for 24 and 48 hours. Decreased cell viability was significant in the cells induced with as little as 32 ng/ml doxycycline in the first 24 hours. Results are presented as fold difference compare to untreated cells at 24 hours. (C) FACS analysis of annexin V/7-AAD stained cells for determination of apoptosis and cell death. Single annexin V positive cells (x-axis, bottom right corner) represent cells undergoing apoptosis, and double positive cells (annexin V⁺ and 7-AAD⁺, right top population) represent dead cells. A slight increase of apoptotic and dead cells was detected at 12 hours which progressed to significant after 24 hours of induction. (D) ATP assay on the cells induced for 24 hours demonstrated that antioxidants (AsAc: ascorbic acid (21.25 mM), B-MET: β-mercaptoethanol (0.5 mM), MTG: monothioglycerol (2.25 mM)) did not have any beneficial effect on cell viability even in cells treated with the low dose of doxycycline (32 ng/ml). (E) Morphology of cells, either uninduced (Control), mDUX-induced (Dox, 125 mg/ml) or induced and treated with antioxidants. (F) Morphology of mDUX inducible fibroblasts (i3T3-mDUX) and inducible mDUX embryonic stem cells (iES-mDUX) (G) after 24 hours of induction with 500 ng/ml doxycyline. mDUX expressed at high levels induces cell death in fibroblasts and embryonic stem cells. (H) ATP assay for effects of doxycycline on viability of control C2C12 and iC2C12 cells after 48 hours of treatment. Results are presented as fold difference compare to untreated C2C12 cells.</p

Pax3 and Pax7 compete with mDUX.

<p>(A) FACS analysis of iC2C12-mDUX cells transduced with MSCV retroviral constructs caring GFP, Pax3-ires-GFP or Pax7-ires-GFP. Almost all of the cells at the time of the experiment stably express GFP (x-axis). (B) Immunofluorescence for Pax3 or Pax7 (red) and GFP (green) reveals that Pax3 and Pax7 are expressed in GFP⁺ cells. Cell number is decreased in induced samples due to toxicity of mDUX. (C) ATP assay for determination of cell viability in iC2C12-mDUX cells transduced with MSCV-ires-GFP, MSCV-Pax3-ires-GFP or MSCV-Pax7-ires-GFP. Cells were induced with various concentrations of doxycycline for 24 and 48 hours. Pax3- and Pax7-expressing cells are largely resistant to the toxicity of mDUX induced by 32 ng/mL dox even after 48 hours of induction. (D) qRT-PCR analyses for MyoD and Myf5 in the cells shown in (C), induced for 18 hours. Expression of MyoD and Myf5 is strongly repressed at 32 ng/mL induction in the control cells, but not the Pax3 or Pax7 expressing cells.</p

mDUX expression in Xenopus.

<p>(A) Neurula stage embryos following injection of <i>GFP</i> mRNA alone (control) or <i>GFP+mDUX</i> mRNA. The green color indicates the domain filled by the RNA injection. The control has a normal neural plate while the mDUX case is very abnormal due to deranged gastrulation movements. (B) Tail muscle pattern in stage 45 tadpoles. The green color shows immunostaining with 12/101 antibody. Control shows the normal pattern of myotomes. Two examples (mDUX) demonstrate how injection into blastomeres V2.1 and 2.2 results in an inhibition of muscle differentiation on the injected (left) side.</p

mDUX and myogenic differentiation.

<p>(A) Phase-contrast microscopy of iC2C12-mDUX cells induced with doxycyline through 6 days of differentiation. (B) Immunofluorescence for detection of MyHC (red, upper panels) and MyoD (red, lower panels) in cells induced with low levels of doxycycline. Nuclei were counterstained with DAPI (blue). iC2C12-mDUX cells were in differentiation medium for 6 days when myotube fusion index was calculated (C). Significantly diminished myogenic differentiation was observed in the cells induced with 10 ng/mL doxycyline. (D) Inhibition of differentiation was confirmed by qRT-PCR. Results are presented as fold difference compared to uninduced cells (0 ng/mL) and the error bars represent the STDEV. (E) Immunofluorescence for detection of MyHC (red) in C2C12 and iC2C12 control cells after 6 days of differentiation and treatment with different concentrations of doxycycline. (F) Calculated fusion index and (G) gene expression analyses of differentiated C2C12 and iC2C12 control cells. Doxycycline by itself did not have any significant effect on myoblast (C2C12 and iC2C12) differentiation.</p

Activity measurements of DUX4 female mice.

<p><b>A)</b> Measurements for various types of activity identifiable in activity monitoring cages. For these studies, N = 6 iDUX4(2.7) females and 6 littermate controls. <b>B)</b> Total ambulatory distance over 24 hours. <b>C)</b> Total ambulatory time over 24 hours. <b>D)</b> Comparison of resting <i>vs</i>. active time. Differences between control and iDUX4 animals were significant to p = 0.014.</p

Additional file 1: Table S1. of DNA-binding sequence specificity of DUX4

Oligos used in this study. (XLS 32.5 KB

Viability of male mice at room temperature.

<p>Viability of male mice at room temperature.</p

The consequence of p53 accumulation in hESCs.

<p>(A) Cell cycle analysis. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) and treated with RA were stained with PI and subjected to flow cytometry analysis to determine DNA content. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#s2" target="_blank">Results</a> quantitated as fold change in cell cycle are shown. (B) qRT-PCR. RNA from hESCs treated with RA for 4 d or Adr for 6 h were subjected to qRT-PCR assay using primers specific for human <i>CDKN1A</i>. (C) ChIP. p53-bound chromatin was immunoprecipitated from hESCs, and p53 enrichment on <i>CDKN1A</i> was analyzed by qRT-PCR using primers encompassing p53REs (*, <i>p</i><0.05). Scheme representing location of p53RE and primers used for ChIP-qRT-PCR are shown on the top (asterisk indicates the 3′ end of the gene). (D) hESCs treated with RA or Adr were lysed, and cell lysates were blotted for γ-H2AX. (E) Apoptosis assay. hESCs treated as in (D) were stained with Annexin V and PI, and percent apoptotic cells was determined by flow cytometry (mean ± SEM). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s004" target="_blank">S4</a>.).</p

Hearing loss in iDUX4 mice.

<p>Auditory brainstem response (ABR) is shown at each frequency indicated over a range of sound levels. N = 5 iDUX4(2.7) and 5 WT littermate control female mice. * p<0.05, **p<0.01.</p