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First published online 21 November 2006
doi: 10.1242/jcs.03290

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PDZRN3 (LNX3, SEMCAP3) is required for the differentiation of C2C12 myoblasts into myotubes

¹ Department of Pharmacology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan
² Department of Surgical Pathology, Yamaguchi University Hospital, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan

View larger version (4K): [in this window] [in a new window]   Fig. 1. Schematic representation of the PDZRN family proteins LNX1 (PDZRN2 or SEMCAP1) and PDZRN3 (LNX3 or SEMCAP3). LNX1 contains an N-terminal RING finger and four PDZ domains, whereas PDZRN3 contains an N-terminal RING finger, two PDZ domains and a consensus-binding motif for class I PDZ domains at its C-terminus. RING-finger and PDZ domains are shown as pink and green boxes, respectively.

View larger version (117K): [in this window] [in a new window]   Fig. 2. Northern blot analysis of the tissue distribution of PZDRN3 mRNA. A nylon membrane containing fractionated polyadenylated RNA from adult human tissues was subjected to hybridization with a [32P]-labeled human PDZRN3 cDNA probe. The arrowhead indicates a hybridizing 5.5 kb mRNA. The blot was also probed for ß-actin mRNA as a loading control. The positions of molecular size standards are indicated on the left. Br, brain; H, heart; Sk, skeletal muscle; Co, colon; Th, thymus; Sp, spleen; K, kidney; Li, liver; In, small intestine; Pl, placenta; Lu, lung; Le, peripheral blood leukocytes.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Abundance of PDZRN3 mRNA in skeletal muscle during postnatal development. (A) Total RNA prepared from skeletal muscle of mice at the indicated ages was subjected to RT-PCR analysis of PDZRN3 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH, control) mRNAs. (B) The amount of PDZRN3 mRNA was quantified by densitometric analysis of gels similar to that shown in (A), normalized by the amount of G3PDH mRNA and expressed as a percentage of the normalized value for day 0. Data are means ± s.e.m. from four independent experiments. *P<0.05 compared with the value for day 0.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Molecular characterization of PDZRN3. (A) Lysates of C2C12 myotubes as well as of HeLa cells infected with an adenovirus containing mouse PDZRN3 cDNA were subjected to immunoblot analysis with polyclonal antibodies to PDZRN3. The positions of molecular size standards are indicated on the left. (B) Lysates of C2C12 myotubes were subjected to immunoprecipitation (IP) with rabbit anti-Semaphorin 4C or control immunoglobulin G (IgG) and the resulting precipitates were subjected to immunoblot analysis with monoclonal antibodies to PDZRN3 or to Semaphorin 4C. Myotube lysates were also subjected directly to immunoblot analysis (Input). (C) GST or GST fusion proteins containing the RING-finger domain of PDZRN3 (GST-PDZRN3-N) or of ARD1 (GST-ARD1-N) were incubated with E1 or E2, as indicated, in a ubiquitination assay. The reaction mixtures were then subjected to immunoblot analysis with anti-ubiquitin (right panel). The purity of the GST and GST fusion proteins used for the ubiquitination assay was evaluated by SDS-PAGE and staining with Coomassie brilliant blue (CBB, left panel).

View larger version (56K): [in this window] [in a new window]   Fig. 5. Up-regulation of PDZRN3 during differentiation of C2C12 myoblasts into myotubes. (A) C2C12 myoblasts induced to differentiate by culture for the indicated times in DM were both examined by phase-contrast microscopy (upper panels) and subjected to immunofluorescence analysis with anti-MHC (lower panels). Bar, 25 µm. (B) C2C12 cell lysates prepared at the indicated times after induction of differentiation were subjected to immunoblot analysis with antibodies to the indicated proteins. (C) The amount of each protein was quantified by image analysis of blots similar to that shown in (B), normalized by the amount of -tubulin and expressed as a percentage of the maximum normalized value in each experiment. Data are means ± s.e.m. from four independent experiments.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Inhibition of myoblast differentiation by depletion of PDZRN3. (A) C2C12 cells were transfected with a control siRNA or an siRNA specific for PDZRN3 mRNA and were induced to differentiate by culture for 4 days in DM. The cells were then examined by phase-contrast microscopy (upper panels) and subjected to immunofluorescence analysis with anti-MHC (lower panels). Bar, 25 µm. (B) Lysates of cells treated as in (A) and cultured in DM for the indicated times were subjected to immunoblot analysis with antibodies to the indicated proteins.

View larger version (54K): [in this window] [in a new window]   Fig. 7. Lack of effect of PDZRN3 depletion on expression of myogenin. (A) C2C12 cells were transfected or not (Mock) with a control siRNA or an siRNA specific for PDZRN3 mRNA and were induced to differentiate by culture for 5 days in DM. They were then examined by phase-contrast microscopy (left panels) and subjected to immunofluorescence analysis with anti-myogenin (middle panels). Nuclei were also stained with 4',6-diamidino-2-phenylindole (DAPI, right panels). Bar, 25 µm. (B) The percentage of DAPI-stained nuclei that were positive for myogenin was determined in each of 10 randomly chosen fields (magnification, 200x) for cells treated as in (A). Data are means ± s.e.m. from a representative experiment.

View larger version (103K): [in this window] [in a new window]   Fig. 8. Expression of PDZRN3 during regeneration of damaged mouse skeletal muscle. (A) Muscle injury and regeneration were induced by intramuscular injection of cardiotoxin in the hind limb and sections of muscle isolated before or 1, 3, 5, or 9 days after injury were stained with hematoxylin-eosin. Bar, 50 µm. (B) Homogenates of muscle isolated at the indicated times after injury were subjected to immunoblot analysis with antibodies to the indicated proteins. The abundance of PDZRN3 was quantified by image analysis of blots similar to that shown in the upper panel and expressed as a percentage of the value at 5 days after injury (lower panel); data are means ± s.e.m. from three independent experiments. (C) A section of muscle at 5 days after injury was double-stained with anti-PDZRN3 (green) and anti-myogenin (red). Myogenin-positive nuclei are indicated by arrows. Myogenin-negative nuclei in myotubes are indicated by arrowheads. Bar, 25 µm.

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