Immunohistochemical profile of liposarcomas.

¤<p>) Desmin and Dlk1 were present in the same cell population.</p>¤¤<p>) In a single location, could be derived from infiltrated muscle.</p>¤¤¤<p>) Dlk1 positive cells correspond with NCAM positive areas.</p

Ectopic ovine Dlk1 inhibits expression of endogenous Dlk1 and affects myostatin expression pattern.

<p>A. Endogenous Dlk1 expression was detected during regeneration using rabbit-anti-murine FA1 antibody and days 3–9 following stab wound are represented. Anti-human FA1 antibody cross-reacts with the ovine form of Dlk1/FA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060692#pone.0060692-Davis1" target="_blank">[8]</a>. Days 3–7 following stab wound are represented. A staining of uninjured LC control muscle confirms that the staining of the ovine form is specific. Scalebar: 50 µm. In LC control mice endogenous Dlk1 is expressed in mononuclear cells located within the injury and primarily observed along the periphery of newly formed myotubes (arrows). In Dlk1 TG mice the expression of Dlk1 is strongly inhibited within the muscle injury and only few mononuclear cells expressing Dlk1 can be observed (arrows). Ectopic ovine Dlk1 is expressed only in the TG mice and not by LC mice. The expression is under control by the myosin light chain promoter and is primarily expressed by myotubes and mature muscle fibers (arrows). B. qPCR analysis of endogenous <i>Dlk1</i> mRNA confirms the down regulation by ectopic ovine Dlk1. Dlk1 was included in the TaqMan™ Low Density Array Card set-up and calculated as described in materials and methods. Black squares: Dlk1 TG mice +/− SD. Open circles: LC control mice. C. RT-PCR analyses of the Dlk1 splice variants present during regeneration. The primers used span exon 5 which contain all splice sites of Dlk1 and theoretically yields bands corresponding to the A (824 bp), B (671 bp), C/C₂ (605/599 bp) and D/D₂ (545/539 bp) forms. Pituitary gland (PG) is used as positive control. This analysis shows that the splice variants present are full-length A, C/C₂ and confirms the down regulation of endogenous Dlk1 by ectopic Dlk, with all splice variants being equally affected. D. Immunohistochemical analysis of myostatin expression in Dlk1 TG and LC mice. Scalebar representative for all images: 20 µm. Myostatin expression is generally patchy and variable both in TG and LC mice, but TG mice present a less intense staining.</p

Immunohistochemical analysis of tumors with adipogenic or osteogenic origin.

<p>A: A biopsy from a liposarcoma with an adipogenic and a myogenic metastasis was investigated for expression of Dlk1, Desmin, Myogenin, Pax7 and NCAM. All myogenic markers were predominantly detected in the myogenic metastasis and not in the main liposarcoma or the adipogenic metastasis. Accordingly, Dlk1 was primarily expressed in the myogenic metastasis. Few Dlk1 positive cells could, however, be detected in the adipogenic metastasis, where few cells were found to express Desmin and Myogenin as well. This supports the observation that Dlk1 is a strong marker for tumors with skeletal muscle phenotype. Scalebar representative for all images: 50 µm. B: Biopsies from tumors with osteogenic origin were analyzed for expression of Dlk1 and all types analyzed (osteosarcoma, osteochondroma and osteoblastoma), were negative for Dlk1 protein expression. Scalebar representative for all images: 50 µm.</p

Dlk1 inhibits myoblast differentiation <i>in vitro</i>.

<p>A: C2C12 cells were constructed to constitutively express full-length Dlk1 under control by the CMV promotor, DLK1-C2C12. Cell morphology and myogenic differentiation potential was analyzed by immunocytochemistry. Forced expression of Dlk1 resulted in inhibition of myogenic differentiation observed as no formation of myotubes, lack of myostatin expression and reduced Desmin staining compared to C2C12 parental control cells. Addition of Dlk1 antibody to the culture supernatant during differentiation reverted the effect of forced Dlk1 expression observed as a regained ability to form myotubes and express myogenin compared to control cells. Scalebars: 20 µm in all images. B: DLK1-C2C12 cells (DLK1) and C2C12 parental control cells were cultured under proliferating conditions for 3 days followed by differentiation for another 5 days. Cells were harvested and analyzed at 1, 3, 5, and 8 days in culture. The differentiation process was analyzed by qPCR of <i>Dlk1</i>, the myogenic markers <i>Mef2a</i>, <i>Myf5</i>, <i>Myod</i>, and <i>Myogenin</i> in addition to <i>Myostatin</i>, <i>Follistatin</i>, <i>TGFβ1</i>, <i>Smad7</i> and <i>Decorin</i>. All qPCR analyses were run in triplicates on duplicate analyses containing triplicate samples (n = 2 for each time point) and presented as relative expression levels with normalization to.<i>Actb</i>, <i>Gusb</i>, <i>Pgk1</i>, <i>Gapdh</i> and <i>Tfrc</i> as described in materials and methods. Black bars: DLK1⁺ cells; white bars: control cells. C: Dlk1 (13 kDa band) and myostatin (a double band corresponding to 40–50 kDa) protein level was analyzed with western blotting during proliferation and differentiation of DLK1-C2C12 and control cells. Dlk1 protein was only expressed by DLK1-C2C12 cells and not by control cells. Myostatin protein was expressed in control cells during differentiation but not during proliferation of either of the cells lines or during differentiation of DLK1-C2C12 cells. However, addition of Dlk1 antibody resulted in expression of myostatin by the DLK1-C2C12 cells during differentiation. Beta-actin (45 kDa) was used as control.</p

Dlk1 expression in osteogenic lesions.

¤<p>After chemo therapy.</p

Dlk1 and myostatin display an inverse relationship in human myoblasts, and the expression of Dlk1 is observed in human rhabdomyosarcoma cell lines.

<p>A: Expression of Dlk1 and myostatin in two human myoblast cultures during G₀ arrest, proliferation before and after G₀, and differentiation. We found that myostatin was highly upregulated during proliferation and differentiation while Dlk1 was downregulated, thus an inverse relation was observed. B. Dlk1 (app. 30 kDa product) and myostatin (a double band around 26 kDa, corresponding to the cleavage product) protein level was analyzed with western blotting during proliferation before and after G₀, during G₀ (cell cycle arrest) and differentiation of primary isolated human muscle cells. Myostatin protein was expressed during proliferation and differentiation but down regulated during G₀. Dlk1 protein was predominantly expressed by G₀ arrested myoblasts. Ponceau Red staining (a common 70 kDa band is shown) confirmed equal loading. C: The expression of Dlk1, Myogenin and Desmin was studied in two human rhabodomyosarcoma cell lines, RD and A204 after induction in differentiation medium. We found that both cultures were Dlk1 and Desmin positive, while only RD cells were Myogenin positive. None of the cultures were able to form myotubes when cultured in differentiation medium. Scalebar representative for all images: 20 µm. D. RT-PCR analysis of Dlk1 splice variants present in RMS cells. The primers used span exon 5 and should thus provide bands for all possible splice variants present. The analysis shows the presence of 3 bands in both RMS cell lines and in the positive control tissue used, placenta and human muscle. These bands correspond to the full length A, and two smaller products most likely the B and C/C2 variants. All bands were sequenced and verified to be Dlk1.</p

Dlk1 transgenic mice display an initially enhanced formation of myotubes following injury.

<p>A morphological analysis of skeletal muscle regeneration in Dlk1 transgenic mice (TG) and littermate control mice (LC) after knife stab injury was performed. Days 3, 4, 5, and 7 post injury is represented for each genotype in a sirius, NCAM (Neural Cell Adhesion Molecule) and dystrophin staining. Dlk1 TG mice display an early presence of newly formed myotubes at day 3 of which most express NCAM and few express dystrophin (arrows). In comparison, the LC control mice only have few myotubes at day 3 as indicated by sirius and NCAM (arrows), and the myotubes have no dystrophin in the membranes until day 5 (arrows). The regenerative response remains accelerated at days 4 and 5 in the Dlk1 TG mice compared to the controls (arrows). Even though the LC control mice display structured muscle tissue at day 5 with expression of both NCAM and dystrophin, the muscle architecture continues to appear more mature in the Dlk1 TG mice at this time point. Scalebar: 30 µm.</p

Data_Sheet_1_CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa.PDF

<p>Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.</p

Immobility-induced skeletal muscle atrophy causes an age-specific decline in muscle size.

<p><b>A.</b> Scheme of experimental setup, including the time points of muscle biopsy procedure. <b>B</b>. Percentage decreases in muscle size (mean muscle fiber area) after 4 days of immobility in young (n = 11) and old (n = 9) as well as after 14 days of immobilization in young (n = 11) and old (n = 12), respectively. * Time effect, p<0.05 compared to pre, # Age effect, p<0.05 young compared to old within time point. Group mean data ± SEM. Mean myofiber area was assessed in the quadriceps femoris muscle, by muscle biopsy sampling. <b>C.</b> Muscle histology from resting state (pre) and immobility (14 d) was analyzed by myofibrillar ATPase at pH 10.3 preincubations demonstrating type I (white) and type II muscle fibers (black) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051238#pone.0051238-Brooke1" target="_blank">[52]</a>.</p

Changes in the transcriptional status of PGC-1α and PGC-1β as a result of immobility-induced disuse-muscle atrophy.

<p>The mRNA level of PGC-1α and PGC-1β was determined using qRT-PCR. <b>A.</b> The results revealed a marked down-regulation of PGC-1α at 24 h in young muscle and a down-regulation in both young and old muscle at the later time points (2 d, 4 d and 14 d). <b>B.</b> The expression levels of PGC-1β mRNA also revealed an age-specific down-regulation after 24 h in young muscle and a down-regulation in both young and old muscle at 2 d and 4 d. In contrast to PGC-1α, the expression levels of PGC-1β mRNA returned to basal levels at 14 days of immobilization, indicating that the two genes may play different roles in the later stages of muscle unloading. * Time effect, p<0.05 compared to pre. Data are geometric means ± back-transformed SEM.</p