Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c

doi: 10.1073/pnas.1613592114

Figure Lengend Snippet: Knockout of Deltex2 accelerates muscle regeneration and enhances satellite cell differentiation capacity. (A) The kinetics of muscle regeneration are enhanced in the absence of Deltex2. Tibialis anterior (TA) muscles of control and Deltex2 KO mice were injured by injection of BaCl2. Cryosections of regenerating muscles were immunostained to detect early regenerating myofibers at 3, 5, 7, and 9 d postinjury, and the numbers of centrally nucleated regenerating fibers were quantified and graphed according to fiber diameter. P < 0.0001 for 3, 5, and 7 d postinjury, χ2 test. (B) The numbers of regenerating myofibers expressing embryonic myosin heavy chain, assessed in the studies described in A. *P < 0.05. (C) Number of Syn4+ve cells counted on single fiber explants from WT and KO (Dtx2−/−) extensor digitorum longus (EDL) muscles immediately after dissociation (day 0) or cultured for 2 d (day 2). The gray line represents the mean. (D and E) Quantitative analysis of the proportion of Syn4+ve SCs expressing MyoD (D) or MyoG (E) per fiber in single fiber explants from WT or Deltex2 KO mice, cultured for 2 d. n = 50. *P < 0.0001.

Article Snippet: MyoD, Myogenin, and Syndecan4 were detected using a mouse monoclonal anti-MyoD antibody (1:1,000; Dako), a mouse monoclonal anti-MyoG antibody (1:250; BD Pharmingen), and a chicken anti-Syndecan4 antibody (1:500), a gift from Dr. Brad Olwin, University of Colorado, for 24 h at 4 °C, followed by 2 h with donkey anti-mouse Alexa Fluor 594 (Invitrogen) and donkey anti-chicken Alexa Fluor 488 (Invitrogen) secondary antibodies.

Techniques: Knock-Out, Cell Differentiation, Injection, Expressing, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c

doi: 10.1073/pnas.1613592114

Figure Lengend Snippet: Multiple sequence alignment of WWE and RING domains in mouse Deltex family members. (A) Mouse Deltex family proteins and RNF146 protein sequences in WWE domains were aligned by a protein sequence alignment program, Clustal Omega (86). The three most conserved amino acid residues in two WWE domains are shown in green boxes. Two amino acid residues, R93 and R170, which are key residues for the binding of the WWE domains to PAR, are highlighted in red boxes. In Mut1, each residue was mutated to disrupt the binding of Deltex2 to PAR. (B) Alignment of protein sequences in RING domains of mouse Deltex family proteins. The key residues to form Zn fingers in the Deltex family RING domains are shown in green boxes. Two amino acid residues, I411 and M454, which are key residues for the interaction of RING domains with E2 ubiquitin-conjugating enzymes, are shown in red boxes. In Mut2, each residue is mutated to R to disrupt the interaction of Deltex2 with its E2 enzyme. (C) Analysis of the levels of Jmjd1c monoubiquitination. (Left) C2C12 myoblasts were transfected with Deltex2 (Dtx2), mutant Deltex2 R93A-R170A (Mut1), or mutant Deltex2 I411R-M454R (Mut2) expression constructs and lysed. Monoubiquitinated Jmjd1c was detected with an anti-ubiquitin antibody (FK2), and total Jmjd1c protein (native and monoubiquitinated) was assessed with an anti-Jmjd1c antibody. (Right) Quantitative analysis of the monoubiquitinated Jmjd1c. n = 3. (D) WT recombinant Deltex2 or two independent activate site mutants were expressed in primary differentiating myoblasts, and MyoD and MyoG mRNA levels were determined by qRT-PCR. *P < 0.05; N.S., not significant.

Article Snippet: MyoD, Myogenin, and Syndecan4 were detected using a mouse monoclonal anti-MyoD antibody (1:1,000; Dako), a mouse monoclonal anti-MyoG antibody (1:250; BD Pharmingen), and a chicken anti-Syndecan4 antibody (1:500), a gift from Dr. Brad Olwin, University of Colorado, for 24 h at 4 °C, followed by 2 h with donkey anti-mouse Alexa Fluor 594 (Invitrogen) and donkey anti-chicken Alexa Fluor 488 (Invitrogen) secondary antibodies.

Techniques: Sequencing, Binding Assay, Transfection, Mutagenesis, Expressing, Construct, Recombinant, Quantitative RT-PCR