Journal: Skeletal Muscle

Article Title: Mesoangioblast delivery of miniagrin ameliorates murine model of merosin-deficient congenital muscular dystrophy type 1A

doi: 10.1186/s13395-015-0055-5

Figure Lengend Snippet: MABs do not synthesize laminin α2 neither in vitro nor in vivo. a Western blot analysis of mouse wild type (C57Bl6) muscle, differentiated and undifferentiated MABs, and medium from differentiated and undifferentiated MABs. Anti-laminin α2 chain antibody detects a specific band only in wild type muscle homogenate, whereas a polyclonal antibody recognizing laminin chain β1 and γ1 detects specific bands in all the homogenates, including medium from differentiated and undifferentiated MABs. Calnexin is shown as loading control (accordingly, it is absent when only medium is loaded). Each lane is loaded with 40 μg of protein. b Cultures of MABs differentiated in myotubes, as depicted by positive staining with anti myosin heavy chain (MyHC) antibody, show positive staining for laminin chains γ1. DAPI staining identifies nuclei. Scale bar = 30 μm. c Cultures of MABs differentiated in myotubes (MyHC positive) do not show staining with anti-laminin α2 chain antibody. DAPI staining identifies nuclei. Scale bar = 30 μm

Article Snippet: Antibodies used for immunohistochemistry and/or Western blot analysis included (p = polyclonal, m = monoclonal, Ch = chicken, Ms = mouse, Rb = rabbit, Gt = goat, Rt = rat): α-actin (mMs Sigma); calnexin (pRb; Sigma C4731); β-dystroglycan (mMs, 8D5, Novocastra); α-dystroglycan (mMs, nIIH6C4, Millipore) and α-dystroglycan (mMs, VIA4-1, Millipore); integrin α5 (pRb, AB1949, Millipore); integrin a6 (mRt, GoH3 from A. Sonnenberg); integrin α7 (pRb, from Dr. U. Mayer); β1 integrin (pRb; Millipore); laminin α1 (mRt, AL-1, Millipore); laminin α2 (mMs; Alexis 4H8-2) and laminin α2 (mMs; 1:200, gift from H. Hori); laminin α4 (pRb, H-194, Santa Cruz); laminin α5 (pRb, 405, from Dr. Sorokin); laminin γ1 (mRt, MAB1914, Millipore); laminin HSA (pRb, L9393, Sigma); myc tag (mMs; Cell Signaling); myosin heavy chain (mMs, Hybridoma Bank); neurofilament M (pCh; 1:1000, Covance PKC-593P); and β-tubulin (mMs; TUB2.1, Sigma).

Techniques: In Vitro, In Vivo, Western Blot, Control, Staining

Journal: Skeletal Muscle

Article Title: Mesoangioblast delivery of miniagrin ameliorates murine model of merosin-deficient congenital muscular dystrophy type 1A

doi: 10.1186/s13395-015-0055-5

Figure Lengend Snippet: MABs can synthesize laminin chains α4 and α5 but not α1. a Cultures of MABs differentiated in myotubes (MyHC positive) do not show staining with anti-laminin α1 chain antibody. DAPI staining identifies nuclei. a′ is the same image as in ( a ), without MyHC staining. Scale bar = 30 μm. b Cultures of MABs differentiated in myotubes show positive staining for laminin α4. DAPI staining identifies nuclei. Scale bar = 30 μm. c Cultures of MABs differentiated in myotubes show positive staining for laminin α5. DAPI staining identifies nuclei. Scale bar = 30 μm

Article Snippet: Antibodies used for immunohistochemistry and/or Western blot analysis included (p = polyclonal, m = monoclonal, Ch = chicken, Ms = mouse, Rb = rabbit, Gt = goat, Rt = rat): α-actin (mMs Sigma); calnexin (pRb; Sigma C4731); β-dystroglycan (mMs, 8D5, Novocastra); α-dystroglycan (mMs, nIIH6C4, Millipore) and α-dystroglycan (mMs, VIA4-1, Millipore); integrin α5 (pRb, AB1949, Millipore); integrin a6 (mRt, GoH3 from A. Sonnenberg); integrin α7 (pRb, from Dr. U. Mayer); β1 integrin (pRb; Millipore); laminin α1 (mRt, AL-1, Millipore); laminin α2 (mMs; Alexis 4H8-2) and laminin α2 (mMs; 1:200, gift from H. Hori); laminin α4 (pRb, H-194, Santa Cruz); laminin α5 (pRb, 405, from Dr. Sorokin); laminin γ1 (mRt, MAB1914, Millipore); laminin HSA (pRb, L9393, Sigma); myc tag (mMs; Cell Signaling); myosin heavy chain (mMs, Hybridoma Bank); neurofilament M (pCh; 1:1000, Covance PKC-593P); and β-tubulin (mMs; TUB2.1, Sigma).

Techniques: Staining

Journal: Skeletal Muscle

Article Title: Mesoangioblast delivery of miniagrin ameliorates murine model of merosin-deficient congenital muscular dystrophy type 1A

doi: 10.1186/s13395-015-0055-5

Figure Lengend Snippet: Human MABs and mouse MABs treated with deacetylating and demethylating substrates express very little amount of laminin α2 chain. a Western blot analysis of homogenates from differentiated mouse MABs (150 μg of protein loaded per lane) untreated (CTR) or treated with 5-aza-2′deoxycytidine ( AZA ), AZA and trichostatin A (AZA + TSA), or trichostatin A ( TSA ) for 3 days. Deacetylation (AZA) and/or demetylation (TSA) do not modify the expression of laminin chain a2, β1, and γ1. Calnexin ( Cnx ) is shown as reference for protein loading. b Quantification of densitometric results of the above the Western blot analysis as ratio between laminin chain α2 and calnexin, representing the mean of three determinations (±SEM). c – d Quantitative PCR analysis for MyoD ( c ) and MyoG ( d ) of differentiated MABs untreated or treated with AZA, AZA + TSA, or TSA; as expected MyoG was significantly increased by AZA and decreased by TSA treatment. e Western blot analysis of homogenates from human fibroblasts, human MABs, and human myoblasts. One hundred micrograms of protein were loaded per lane. Anti-laminin α2 chain antibody detects positive band in fibroblasts and myoblasts, whereas very little (if any) positive staining is present in human MABs. All three homogenates stained positive for laminin chain β1 and γ1

Article Snippet: Antibodies used for immunohistochemistry and/or Western blot analysis included (p = polyclonal, m = monoclonal, Ch = chicken, Ms = mouse, Rb = rabbit, Gt = goat, Rt = rat): α-actin (mMs Sigma); calnexin (pRb; Sigma C4731); β-dystroglycan (mMs, 8D5, Novocastra); α-dystroglycan (mMs, nIIH6C4, Millipore) and α-dystroglycan (mMs, VIA4-1, Millipore); integrin α5 (pRb, AB1949, Millipore); integrin a6 (mRt, GoH3 from A. Sonnenberg); integrin α7 (pRb, from Dr. U. Mayer); β1 integrin (pRb; Millipore); laminin α1 (mRt, AL-1, Millipore); laminin α2 (mMs; Alexis 4H8-2) and laminin α2 (mMs; 1:200, gift from H. Hori); laminin α4 (pRb, H-194, Santa Cruz); laminin α5 (pRb, 405, from Dr. Sorokin); laminin γ1 (mRt, MAB1914, Millipore); laminin HSA (pRb, L9393, Sigma); myc tag (mMs; Cell Signaling); myosin heavy chain (mMs, Hybridoma Bank); neurofilament M (pCh; 1:1000, Covance PKC-593P); and β-tubulin (mMs; TUB2.1, Sigma).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: Skeletal Muscle

Article Title: Mesoangioblast delivery of miniagrin ameliorates murine model of merosin-deficient congenital muscular dystrophy type 1A

doi: 10.1186/s13395-015-0055-5

Figure Lengend Snippet: MABs + mMAG can express and synthesize miniagrin and when injected in dy 2J colonize the skeletal muscles but not the peripheral nerves. a C57 MABs transduced with miniagrin gene carrying myc tag show diffuse positivity when stained with anti-myc antibody ( a DAPI staining identifies nuclei); scale bar = 30 μm. b Western blot analysis with anti-myc antibody, recognizing mMAG, of cell homogenate and medium from cultures of C57 MABs and C57 MABs transduced with miniagrin gene (MABs + mMAG). Anti-myc recognizes a band only in the homogenate and medium of transduced MABs. β-Tubulin is shown as loading control (accordingly, staining is not present in medium). Each lane is loaded with 25 μg of proteins. b Cryosection of the tibialis anterior muscle from dy 2J mouse treated for 10 days with MABs + mMAG. Double staining with anti-laminin γ1 ( a depicting myofibers, in red ) and anti-myc antibody ( b recognizing mMAG, in green ) shows diffuse expression of miniagrin around myofibers; scale bar = 50 μm. c Same muscle showing myc-positive fibers with centrally located nuclei (identified by DAPI, in blue ), suggesting regenerating fibers; scale bar = 25 μm. c Cryosection of the tibialis anterior muscle from dy 2J mouse treated for 10 days with MABs + mMAG. Double staining with anti-laminin γ1 ( a depicting myofibers, in red ) and anti-myc antibody ( b recognizing mMAG, in green ) shows that myc positivity is not diffuse to the entire muscle but limited to the area of injection ( asterisk ). Scale bar = 50 μm. d Cryosection of the tibialis anterior muscle from dy 3K mouse 10 days after i.m. injection of MABs carrying a myc tag. Staining with anti-myc antibody recognizes an area where MABs secreted mMAG a , whereas staining with anti-laminin α2 antibody does not show positive signal ( b DAPI staining identifies nuclei). Scale bar = 25 μm. e Cryosection of intramuscular nerves of the tibialis anterior muscle from dy 2J mouse treated for 10 days with MABs + mMAG. Double staining with neurofilaments ( NF ) recognizing axons a and anti-myc antibody b recognizing mMAG. Myc staining is restricted to the perineurium and blood vessels, whereas only scarce and scattered positivity is present in the endoneurium. DAPI staining identifies nuclei. Scale bar = 20 μm

Article Snippet: Antibodies used for immunohistochemistry and/or Western blot analysis included (p = polyclonal, m = monoclonal, Ch = chicken, Ms = mouse, Rb = rabbit, Gt = goat, Rt = rat): α-actin (mMs Sigma); calnexin (pRb; Sigma C4731); β-dystroglycan (mMs, 8D5, Novocastra); α-dystroglycan (mMs, nIIH6C4, Millipore) and α-dystroglycan (mMs, VIA4-1, Millipore); integrin α5 (pRb, AB1949, Millipore); integrin a6 (mRt, GoH3 from A. Sonnenberg); integrin α7 (pRb, from Dr. U. Mayer); β1 integrin (pRb; Millipore); laminin α1 (mRt, AL-1, Millipore); laminin α2 (mMs; Alexis 4H8-2) and laminin α2 (mMs; 1:200, gift from H. Hori); laminin α4 (pRb, H-194, Santa Cruz); laminin α5 (pRb, 405, from Dr. Sorokin); laminin γ1 (mRt, MAB1914, Millipore); laminin HSA (pRb, L9393, Sigma); myc tag (mMs; Cell Signaling); myosin heavy chain (mMs, Hybridoma Bank); neurofilament M (pCh; 1:1000, Covance PKC-593P); and β-tubulin (mMs; TUB2.1, Sigma).

Techniques: Injection, Muscles, Transduction, Staining, Western Blot, Control, Double Staining, Expressing