DOR loss-of-function alters myogenesis.

<p>Panels A–F. Confluent C2C12 myoblasts previously infected with lentiviruses encoding scrambled RNA (squares) or DOR siRNA (triangles) were allowed to differentiate in 5% horse serum-containing medium for 4 days. Total RNA was purified and the expression of <i>DOR</i>, <i>myogenin</i>, <i>caveolin-3</i>, <i>actin α1</i>, <i>creatine kinase</i>, <i>IGF-II</i> and <i>HPRT</i> was assayed by real-time PCR. Values were expressed as relative to <i>HPRT</i>. Results are mean±SD of four independent experiments. Scrambled and DOR siRNA groups were significantly different as analyzed by two-way ANOVA, at P<0.05. Panel G. DOR and muscle-specific protein expression (myogenin, caveolin 3, and glycogen synthase) were analyzed by Western blot of total cell lysates (20 µg) from each condition. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein β-actin.</p

DOR rapidly delocalizes from PML nuclear bodies in response to thyroid hormones.

<p>Panel A. HeLa cells were transiently cotransfected with DOR and TR_α1 expression vectors. The intranuclear positioning of DOR relative to PML nuclear bodies was determined before and after T₃ addition. Antibodies and immunofluorescence legend: Anti-DOR, stained red (column 1); anti-PML, stained green (column 2); merged images (column 3). Panel B. Full length DOR was fused in frame with the fluorescent protein GFP. To determine whether DOR-GFP retained the capacity to coactivate TR_α1, experiments were done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001183#pone-0001183-g003" target="_blank">Figure 3A</a>. * significant difference compared to the nuclear hormone receptor group, at P<0.05 (post hoc <i>t</i> test). Panel C. HeLa cells were transiently cotransfected with DOR-GFP and TR_α1expression vectors. The intranuclear positioning of DOR relative to PML nuclear bodies and TR_α1was determined before and after a range of times after T₃ addition. Antibodies and immunofluorescence legend: Anti-DOR, stained red (column 1); anti-PML, stained green (column 2), anti-TR_α1 cyan. Merged images: DOR/PML (column 3), DOR/TR_α1 (column 4).</p

DOR transactivates nuclear hormone receptors.

<p>Panel A. HeLa cells were transfected with expression plasmids encoding TR_α1 (TR), DOR, the empty vector pcDNA3 as a control vector, and the reporter vectors containing TR_α1 response elements linked to CAT. Cells were treated for 18 h in the presence or absence of ligands (100 nM T₃) and assayed for reporter expression. Results are mean±SD of 6 independent experiments. * significant difference compared to the nuclear hormone receptor group, at P<0.05 (post hoc <i>t</i> test). Panel B. Reporter assays were done as in previous panels but the amounts of DOR (ranging from 200 to 600 ng) used for transfection differed and these assay were done in the presence of ligands. Results are mean±SD of 6 independent experiments. * significant difference compared to the nuclear hormone receptor group, at P<0.05 (post hoc <i>t</i> test).</p

DOR shows transcriptional activity when tethered to a target gene promoter.

<p>DOR or fragments corresponding to the amino acids indicated were fused to the DNA-binding domain of Gal4 (Gal4 DBD) and transfected in HeLa cells (panel A) or in HEK293T cells (panel B). Transcription was assayed with a reporter plasmid containing five copies of the UAS linked to luciferase. Results are mean±SD of 6 independent experiments. * difference compared to the Gal4 DBD-DOR group, at P<0.05 (post hoc <i>t</i> test).</p

DOR loss-of-function in muscle cells.

<p>Panel A. C2C12 myoblasts previously infected with lentiviruses encoding scrambled RNA (open bar) or DOR siRNA (black bar) were cultured. Cell extracts and total RNA were obtained and DOR protein and mRNA levels were assayed by Western blot and real-time PCR. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein β-actin. * difference compared to the scrambled group, at P<0.05 (Student's <i>t</i> test). Panel B. Scramble (open bars) or DOR siRNA C2C12 muscle cells (black bars) were transfected with a reporter vector driven by a TRE, and with or without a expression vector for TR_α1. Cells were then incubated in the presence or absence of thyroid hormone for 16 h. Results are mean±SD of triplicates and are representative of three independent experiments. * difference compared to the scrambled group, at P<0.05 (post hoc <i>t</i> test). Panels C–H. Scrambled (open bars) or DOR siRNA C2C12 muscle cells (black bars) were incubated in 5% horse serum-containing medium either in the absence or in the presence of 100 nM T₃. Total RNA obtained at 48 h of T_{3 t} treatment were assayed by real-time PCR to measure the expression of several genes. Results are mean±SD of a representative experiment. * difference compared to the control group, at P<0.05 (post hoc <i>t</i> test).</p

DOR binds <i>in vitro</i> and <i>in vivo</i> to thyroid hormone receptors.

<p>Panel A. GST protein or TR_α1 fused to GST (TR-GST) were immobilized on glutathione sepharose beads and incubated with the DOR protein containing an N-terminal histidine tag (HisDOR), with or without the ligand (1 µM T₃). Bound proteins were eluted and resolved by SDS-PAGE and further Western blot using an antibody against the histidine tag (to visualize HisDOR) or against GST (to visualize GST or TR-GST). Panel B. HeLa cells over-expressing His-tagged DOR (left), TRα-1 (middle), or both (HisDOR+TR_α1) (right) were exposed to T₃ or were left untreated. After 1 h of treatment, cells were collected and DOR was immunoprecipitated from the nuclear fractions. The input control (10% input) and the immunoprecipitates (IP) were assayed by Western blot with specific antibodies. Panel C. ChIP analysis over a T₃ responsive promoter. DOR and TR_α1-transfected HeLa cells were treated with T₃ for 1 h or left untreated. Cross-linked chromatin prepared from cells was immunoprecipitated with the antibodies indicated. As negative controls, the samples were subjected to ChIP in the absence of antibody or in the presence of an irrelevant antibody (anti-hemaglutinin, HA). Aliquots of chromatin taken before immunoprecipitation (input) and the immunoprecipitates were subjected to PCR analysis with primers directed to the dio1 promoter. DOR immunoprecipitates were used to amplify <i>IL-2</i> (an additional negative control group).</p

DOR sequences and tissue distribution of <i>DOR</i> expression and down-regulation in skeletal muscle from ZDF rats.

<p>Panel A. Amino acid sequence of human, mouse and rat DOR proteins (sequences 1, 2 and 3, respectively). Multi-alignment done using the CLUSTALW Sequence Alignment programme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001183#pone.0001183-Thompson1" target="_blank">[52]</a>. Amino acids differing from the consensus are inverse. The amino acid residues used to generate the polyclonal antibodies are shown in bold. The C-terminal basic motif, indicated by a line of “+”, is predicted to form an alpha-helix structure whereas the N-terminal half is unstructured (GLOBPLOT 2). Panel B. PolyA⁺-RNA membrane containing human adult tissues was probed with ³²P-labelled rat <i>DOR</i> cDNA and washed in stringent conditions. The probe hybridises to a transcript of approximately 4.5 kb. Hybridisation with human glycerol-3-phosphate dehydrogenase (GPDH) cDNA was used as a control probe. Br, brain; He, heart; SK, skeletal muscle; Co, colon; Th, thymus; Sp, spleen; Ki, kidney; Li, liver; Sl, small intestine; Pl, placenta; Lu, lung; Leu, leukocytes. Panels C. Total RNA was purified from several rat tissues and subjected to Northern blot analysis. Ethidium bromide staining of the ribosomal 28S subunit was used as a control of the relative amounts of RNA loaded in each lane and to check the integrity of RNA in each sample. SK, skeletal muscle; He, heart; WAT, white adipose tissue; Ki, kidney; Br, brain; Lu, lung. Panel D. Total RNA was purified from skeletal muscle from non-diabetic and ZDF rats, and RNA was subjected to Northern blot analysis. The mean±SD of 6 separate observations is shown. * difference compared to the control group, at P<0.01 (Student's <i>t</i> test).</p

Analysis of body weight, contents of DNA, RNA and protein in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 5).

<p>a,b - different superscripts indicate significant differences (p<0.05);</p>*<p>- significantly different if compared to 11-week DU6P or Fzt:DU, respectively as evaluated using the Wilcoxon-signed rank test.</p><p>Furthermore the non-polysomal and polysomal RNA fraction in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 4) was analysed.</p

In mid-aged female DU6P mice inhibition of PTEN by means of reduced PTEN expression and increased PTEN phosphorylation correlates with higher levels of AKT phosphorylation at Ser-473, inactivation of GSK3ß on one hand and higher levels of S6K on the other.

<p>An increase of muscle mass is due to higher levels of muscle glycogen and an increased rate of protein synthesis in female 29-week DU6P mice.</p

Analysis of signal transduction in muscle lysates from 11, 29 and 54-week female DU6P and Fzt:DU mice.

<p>Western blot identified phosphorylated and total expression of the respective signaling molecule. Specific activation was calculated from the ratios of phosphorylated versus total protein. Coomassie blue staining of the membranes used for Western immuno detection was used as loading control. A: IGF-1Rß in membrane fractions (n = 3); B: ILK (n = 9); C: GDF-8, 26 kDa band (n = 11) and D: PTEN (n = 9). The inserts provide representative experiments, whereby all samples were studied on the identical membrane. Sample numbers (n) depict the number of samples per age group, the error bars represent SEM.</p