Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 1. Gene and protein expression of LKB1 in GC muscles of all experimental groups. (A-D) RT-PCR and western blot (WB) analyses of LKB1 expression in the following mice: (A) C57BL/10 mdx mice (n=10) versus C57BL/10 WT mice (n=11) (*P<0.036); (B) D2 mdx mice (n=12) versus D2 WT mice (n=9) (*P<0.0192); (C) C57BL/10 mdx exercised (EXER) mice (n=4) versus C57BL/10 mdx sedentary (SED) mice (n=4) (*P=0.0153); (D) C57BL/10 mdx mice treated with metformin (MET) (n=4) versus C57BL/10 mdx mice (n=4) (*P=0.015). Blots were loaded by an operator unaware of the experimental groupings of samples. In A, the most representative blot of the three western blot experiments performed is shown because of the large number of samples belonging to Group 1. Data are mean+s.e.m. Significant differences were assessed by unpaired Student’s t-test.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Expressing, Muscles, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 2. Immunofluorescence imaging for LKB1. GC muscle sections of 6-month-old BL10 mdx and WT mice were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown at the bottom.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 7. Gene expression and localization of LKB1 in WT and dystrophic murine muscle cells. (A) Lkb1 expression in 2B4 (WT) and SF1 (DMD) myoblasts (D0), and myocytes (D2-D11). Significant differences were assessed by unpaired Student’s t-test (*P≤0.05; ***P≤0.001). ns, not significant. (B) Immunofluorescence imaging for LKB1. D11 2B4 and SF1 cells were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown on the right.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Gene Expression, Expressing, Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 8. Schematic summarizing the pathways involved in the LKB1- AMPK-HDAC axis in healthy and dystrophic muscle. In healthy muscle, LKB1 is activated in response to exercise, in turn stimulating the metabolic remodeling mediated by the key modulator AMPK. AMPK turns off the class II HDACs that are also inhibited via SIK phosphorylation by LKB1. In dystrophic muscle, this refined mechanism of regulation is altered owing to LKB1 impairment. This allows class II HDACs to carry on their epigenetic silencer action, thus inhibiting the expression of genes involved in oxidative metabolism (Mef2c).

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Phospho-proteomics, Expressing

Journal:

Article Title: Arsenic Stimulates Sinusoidal Endothelial Cell Capillarization and Vessel Remodeling in Mouse Liver

doi: 10.1002/hep.21444

Figure Lengend Snippet: Antibodies Used in Immunofluorescence and Western Analysis

Article Snippet: Fluorescent images were captured with an Olympus Fluoview 500 confocal microscope (Malvern, NY) or a Nikon microphot-FXL microscope, fitted with an Olympus CCD digital camera. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Protocol 1° Antibody Source (Dilution) 2° Antibody Source/Fluorophor (Dilution) PECAM (CD31) IF Monoclonal rat anti-mouse CD31 (clone-MEC13.3) BD Biosciences, San Jose, CA (1:100) Goat anti-rat Alexa 488, Invitrogen, Carlsbad, CA (1:500) Laminin-1 IF Polyclonal rabbit anti-laminin Sigma, St. Louis, MO (1:750) Goat anti-rabbit Alexa 594, Invitrogen (1:500) Caveolin-1 IF Polyclonal rabbit anti-caveolin-1, Cell Signaling Technologies, Danvers, MA (1:300) Goat anti-rabbit Alexa 594, Invitrogen (1:500) αSMA IF Monoclonal anti-alpha smooth muscle actin (clone 1A4), Sigma (1:500) Goat anti-mouse Alexa 594, Invitrogen (1:500) Actin WB Monoclonal mouse anti-actin (clone C4) Chemicon, Temecula, CA (1:1000) Sheep anti-mouse HRP, GE Healthcare, Piscataway, NJ (1:5000) Rac1 WB Monoclonal mouse anti-rac1 (clone-102), BD Biosciences (1:1000) Sheep anti-mouse HRP, GE Healthcare (1:5000) Nucleus IF Not applicable DRAQ5, Biostatus, Leicestershire, UK (1:2000) Open in a separate window Abbreviations: IF, immunofluorescence; WB, western blot.

Techniques: Immunofluorescence, Western Blot

Journal: The Journal of Cell Biology

Article Title: Megf10 regulates the progression of the satellite cell myogenic program

doi: 10.1083/jcb.200709083

Figure Lengend Snippet: Megf10 is expressed in quiescent and activated satellite cells. (A) Immunohistochemistry on frozen sections of tibialis anterior muscles from 2-mo-old mice revealed that Megf10 was coexpressed with syndecan 4 in resting skeletal muscle. Arrowheads point to double-positive cells. Bar, 10 μm. (B) Individual myofibers isolated from the EDL muscle of adult mice were costained for Megf10, Pax7, and syndecan 4 or M-cadherin. Megf10 expression was limited to cells that also expressed Pax7 and syndecan 4 or M-cadherin (arrowheads), demonstrating that Megf10 expression is limited to quiescent satellite cells in resting skeletal muscle. Bar, 10 μm. (C) Individual fibers from the EDL muscle of Myf5-Cre*ROSA-YFP reporter mice were isolated, freshly fixed, and stained for YFP, Pax7, and Megf10 expression. Megf10 expression was detected in Pax7 + /YFP + quiescent satellite cells but never in Pax7 + /YFP − quiescent satellite cells. Arrow points to a Pax7 + /YFP − /Megf10 − satellite cell. (D) Myofibers were cultured for 3 d and stained for Pax7, MyoD, and myogenin in the same color to label all satellite cells. Counterstaining with the Megf10 antibody demonstrated that all (99.5%; n > 1,000) activated satellite cells express Megf10. Bar, 50 μm. (E) The presence of a Pax7 + /YFP + /Megf10 + progenitor cell (arrowhead) and a Pax7 + /YFP − /Megf10 − stem cell (arrow) on the same fiber after isolation. After 3 d in culture, both Pax7 + /YFP − (arrow) and Pax7 + /YFP + (arrowhead) proliferating cells express Megf10. Bars, 50 μm.

Article Snippet: Antibodies used for these studies were as follows: α-MyoD (C-20; Santa Cruz Biotechnology, Inc.), α-Myf5 (C-20; Santa Cruz Biotechnology, Inc.), α-Pax7 (PAX7; Developmental Studies Hybridoma Bank), α-HA (HA7; Sigma-Aldrich), α-myogenin (F5D [Developmental Studies Hybridoma Bank] or M225 [Santa Cruz Biotechnology, Inc.]), and α-MyHC (MF20; Developmental Studies Hybridoma Bank). α-Megf10 antibody was generated in rabbits against a GST fusion protein containing the carboxy-terminal 290 aa of Megf10 (Washington Biotechnology).

Techniques: Immunohistochemistry, Muscles, Isolation, Expressing, Staining, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Megf10 regulates the progression of the satellite cell myogenic program

doi: 10.1083/jcb.200709083

Figure Lengend Snippet: Overexpression of Megf10 alters the levels of key myogenic proteins. (A) Western blot of protein extracts from Megf10-overexpressing cells and empty vector puro controls. Myf5 levels are elevated in Megf10-overexpressing cells, whereas MyoD and Pax7 are down-regulated under growth conditions. Cells overexpressing Megf10 fail to up-regulate myogenin during differentiation. (B) Quantitative PCR analysis reveals that although Myf5 and Pax7 RNA levels are altered in Megf10-expressing cells, alterations in MyoD are occurring mainly at the protein level (*, P < 0.02). Transcript levels are normalized to GAPDH and control levels are set to 1 ( n = 3). Error bars represent SEM.

Article Snippet: Antibodies used for these studies were as follows: α-MyoD (C-20; Santa Cruz Biotechnology, Inc.), α-Myf5 (C-20; Santa Cruz Biotechnology, Inc.), α-Pax7 (PAX7; Developmental Studies Hybridoma Bank), α-HA (HA7; Sigma-Aldrich), α-myogenin (F5D [Developmental Studies Hybridoma Bank] or M225 [Santa Cruz Biotechnology, Inc.]), and α-MyHC (MF20; Developmental Studies Hybridoma Bank). α-Megf10 antibody was generated in rabbits against a GST fusion protein containing the carboxy-terminal 290 aa of Megf10 (Washington Biotechnology).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: The Journal of Cell Biology

Article Title: Megf10 regulates the progression of the satellite cell myogenic program

doi: 10.1083/jcb.200709083

Figure Lengend Snippet: Megf10 regulates satellite cell activation and proliferation. (A) Individual fibers isolated from EDL muscles were transfected with Megf10 siRNA, cultured for 3 d, and stained for Pax7 and Megf10. siRNA knockdown induces a loss of Megf10 protein expression by the majority of the activated satellite cells ( n = 3). Bar, 50 μm. (B) Histograms depicting individual counts of the total numbers of activated satellite cells (labeled with Pax7 and MyoD/myogenin) on fibers transfected with Megf10 siRNA. The mean number of myogenic cells per fiber is reduced by a mean of 30% as compared with scrambled siRNA controls ( n = 3). (C) Knockdown of Megf10 dramatically reduces the number of self-renewing progenitors (Pax7 + /MyoD − ) from 19 to 7% and respectively increases the number of Pax7 + /MyoD + cells ( n = 3; **, P = 0.008). Error bars represent SEM. (D) Although the number of activated progenitor cells is reduced, the overall ratio of stem cell (YFP − ) to progenitor cell (YFP + ) remains unchanged ( n = 3). Error bars represent SEM.

Article Snippet: Antibodies used for these studies were as follows: α-MyoD (C-20; Santa Cruz Biotechnology, Inc.), α-Myf5 (C-20; Santa Cruz Biotechnology, Inc.), α-Pax7 (PAX7; Developmental Studies Hybridoma Bank), α-HA (HA7; Sigma-Aldrich), α-myogenin (F5D [Developmental Studies Hybridoma Bank] or M225 [Santa Cruz Biotechnology, Inc.]), and α-MyHC (MF20; Developmental Studies Hybridoma Bank). α-Megf10 antibody was generated in rabbits against a GST fusion protein containing the carboxy-terminal 290 aa of Megf10 (Washington Biotechnology).

Techniques: Activation Assay, Isolation, Muscles, Transfection, Cell Culture, Staining, Knockdown, Expressing, Labeling

Journal: The Journal of Cell Biology

Article Title: Megf10 regulates the progression of the satellite cell myogenic program

doi: 10.1083/jcb.200709083

Figure Lengend Snippet: Loss of Megf10 results in precocious differentiation. (A) EDL fibers were transfected with Megf10 siRNA, cultured for 60 h, and stained for Pax7 and myogenin. Megf10 knockdown induces three times more cells to undergo precocious differentiation ( n = 3; **, P = 0.004). Error bars represent SEM. Bar, 50 μm. (B) Quantitative PCR analysis demonstrates up-regulation of myogenin and MyHC and down-regulation of Pax7, MyoD, and Myf5 in wild-type primary myoblasts in which Megf10 has been knocked down ( n = 6; *, P < 0.05; **, P < 0.01). Error bars represent SEM. (C) Quantitative PCR demonstrates down-regulation of components of the Notch signaling pathway after shRNA knockdown of Megf10 in MyoD − / − primary myoblasts ( n = 3; *, P < 0.05; **, P < 0.01). Error bars represent SEM.

Article Snippet: Antibodies used for these studies were as follows: α-MyoD (C-20; Santa Cruz Biotechnology, Inc.), α-Myf5 (C-20; Santa Cruz Biotechnology, Inc.), α-Pax7 (PAX7; Developmental Studies Hybridoma Bank), α-HA (HA7; Sigma-Aldrich), α-myogenin (F5D [Developmental Studies Hybridoma Bank] or M225 [Santa Cruz Biotechnology, Inc.]), and α-MyHC (MF20; Developmental Studies Hybridoma Bank). α-Megf10 antibody was generated in rabbits against a GST fusion protein containing the carboxy-terminal 290 aa of Megf10 (Washington Biotechnology).

Techniques: Transfection, Cell Culture, Staining, Knockdown, Real-time Polymerase Chain Reaction, shRNA