List of primers used for RT–PCR splicing analysis.

<p>Name of genes, exons analyzed and primer sequences are indicated. RT-PCR analysis were performed in Muscle (M), Heart (H) or both (M/H).</p

Proteasome activity in DMSXL mice.

<p>Chymotrypsin-like activities of the proteasome were assayed using the fluorogenic peptides LLVY-AMC in 4-month-old female WT and DMSXL mice. Each group contained ten animals and each animal was assayed in triplicate. Histogram bars represent the average activity ± SD for each group (* p<0.05, Student's t test).</p

Histological abnormalities in DMSXL muscles.

<p>(A) Fiber size (cross-sectional area, pixels) was measured in tibialis anterior (TA) and soleus muscles from 4-month-old female WT (grey line) and DMSXL (black line) mice, n = 3 biological replicates in both groups. (B) Fiber types were determined in TA and soleus muscles from WT (grey histograms) and DMSXL (black histograms) mice, n = 3 biological replicates in both groups. (C) Oil Red O staining of muscles section (TA) showed intramyocellular lipid accumulation in DMSXL mice. Data are presented as means ± SEM (* p<0.05, ** p<0.01, *** p<0.001, Chi-square or Student's t tests), n = 4–5 in both groups.</p

Foci distribution in mice carrying CTG expansions.

<p>The distribution and quantification of <i>DMPK</i> RNA foci in various tissues (A) and muscles (B) were compared in 8 month-old homozygous mice with 500/600 CTG (DM300) or 1000/1400 CTG (DMSXL). HL, hind leg; FL foreleg. n is for the number of foci per nucleus. LF: large foci.</p

Schematic representation of the <i>DMPK/SIX5</i> region.

<p>Dashed lines represent sense and antisense <i>DMPK</i> transcript. A, B′, C and C′ represent the localization of amplicons used for qRT-PCR. CTCF bs: CTCF binding site; Enh/pro enhancer and promoter region. pA: polyadenylation site for the <i>DMPK</i> sense mRNA The <i>SIX5</i> start site is indicated with an arrow. The location of the CpG island is indicated in grey.</p

Motor performance of DMSXL mice.

<p>(A) Treadmill test was performed on WT and DMSXL female mice between 4 and 6 months of age. The Critical Speed was calculated from the slope of the regression line, plotting the distance versus the time to exhaustion, n = 9 for both groups. (B) The wheel running test was performed on WT and DMSXL mice between 4 and 6 months of age. The mice were followed over a period of 10 days. The distance, the maximal speed and the time spent running are shown, n = 14 biological replicates for both groups. Data are presented as means ± SEM (* p<0.05, ** p<0.01, Student's t test or Mann-Whitney test).</p

Circulating levels of growth-related signaling proteins after fasting.

<p>Significant decreases of IGFBP-3 and insulin levels were observed in 4-month-old homozygous DMSXL. A decrease of IGF-I was also observed but did not reach statistical significance. GH levels were variable but similar between DMSXL and WT. Data are presented as means ± SEM (* p<0.05, ** p<0.01, Student's t test or Mann-Whitney test), n = 12 biological replicates per group.</p

Foci accumulation was detected for both sense and antisense <i>DMPK</i> transcripts.

<p>FISH experiments using 5′-Cy3- labeled (CAG)5 or 5′-Cy3- labeled (CTG)5 probes detected respectively, in red, sense and antisense RNA foci in heart (A) and muscle (B) from DMSXL homozygote.</p

Splicing deregulation in DMSXL mice.

<p>Percentages of alternative exon inclusion in mRNA transcripts were studied by RT-PCR in 2- and 4-month-old mice in tibialis anterior (A–B) and heart (C–D), and in various muscles from 2-month-old mice (E). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003043#s2" target="_blank">Results</a> were compared between DMSXL (n = 6) and WT (n = 6). Each bar represents the mean of 6 biological replicates ± SEM. Differences in percentage of alternative exon inclusion between DMSXL and WT mice were determined to be statistically significant by Student's t test, * p<0.05, ** p<0.01, *** p<0.001. 2 m, 2- month-old; 4 m, 4-month-old.</p

<i>DMPK</i> expression profiles.

<p>Expression of the human <i>DMPK</i> transgene was studied in various hemizygous DMSXL tissues (A) and muscles (B), in parallel with the endogenous <i>Dmpk</i> mouse gene (C and D) (n = 3). (E) Expression of <i>DMPK</i> in human tissues. Dia., Diaphragm; Sterno., Sternomastoid; Quadri, Quadriceps; TA, Tibialis Anterior; Gastroc. Gastrocnemius. (a.u.): arbitrary units. (F) Expression of <i>DMPK</i> in hemizygous (Hemi.) and homozygous (Homo.) DMSXL tissues. Data are presented as means ± standard deviation.</p