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

Modular transcriptional fingerprint for complete KD in the training, test and incomplete KD sets.

<p>Colored dots represent the percentage of significantly overexpressed (red) or underexpressed (blue) transcripts within a module in patients with complete KD compared to controls (see [D] for module map key). Blank modules indicate no significant differences between patients and controls. (A) Patients with complete KD demonstrated significant overexpression of modules related to inflammation (M3.2, M4.2, M4.6, M4.13, M5.1, 5.7), platelets (M1.1), neutrophils (M5.15). Conversely, genes related to adaptive immunity: T cells (M4.1, M4.15), B cells (M4.10), lymphoid lineage (M6.19), and cytotoxicity/NK cells (M3.6), were significantly underexpressed. (D) Key to the functional interpretation of each transcriptional module (M): module sets 1 to 6 are indicated on the y-axis, and module numbers within each set are indicated on the x-axis. Scatter plot showing modular correlations (Spearman's r) between training (x-axis) and the test (y-axis) sets (E) and incomplete KD set (F). The inflammation modules (M4.6, M4.2, 3.2) were the most highly correlated between the training and the test sets.</p

Multivariable logistic regression for the outcome of non-response to treatment.

<p>Multivariable logistic regression for the outcome of non-response to treatment.</p

Demographic, clinical and laboratory data of study patients.

<p>Demographic, clinical and laboratory data of study patients.</p

Transcriptional profiles in Kawasaki disease versus adenovirus and Group A streptococcus infections.

<p>Supervised learning KNN algorithm with 6 neighbors and a p value ratio cutoff of 0.5 was used to identify the top genes that best discriminated cKD from adenovirus (AdV) disease from (A) and GAS (B). Predicted class is indicated by the corresponding lighter-colored rectangles. <b>(A)</b> The KNN algorithm identified 25 classifier genes that best discriminated AdV from KD. Leave one out cross validation of the training set (A) and test set (B) correctly classified 43 of 45 patients with 96% accuracy (complete KD [n = 26; blue); AdV [n = 19, red]. <b>(B)</b> Using the same strategy we identified 10 genes that best discriminated GAS from cKD. (C and D). Leave-one-out cross-validation of the 10 classifiers in the training set (C) and test set (D) correctly classified 28 of the 36 samples (complete KD [n = 19; blue]; GAS [n = 17, green] with 78% accuracy; one patient in the training set and test sets could not be predicted.</p

Biosignature of KD.

<p><b>(</b>A) Statistical group comparisons between children with complete KD and healthy matched controls (HC) (Mann-Whitney test p<0.01, Benjamini-Hochberg multiple test correction and 1.25-fold change) yielded 8,799 significantly differentially expressed transcripts. Transcripts were organized by hierarchical clustering, where each row represents a single transcript and each column an individual participant. Normalized expression levels are indicated as overexpressed (red) or underexpressed (blue) compared to the median expression of healthy controls (yellow). (B) The 8,799 biosignature was applied to an independent test set of 37 children with complete KD and HC. Unsupervised hierarchical clustering of the KD signature grouped all complete KD patients in the test set together (blue bar) except for one patient who was on the ninth day of illness at the time of sample collection, and (C) all incomplete KD patients were grouped together. Black line indicates the cluster separation.</p