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1.
Fig. 4.

Fig. 4. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Laminin-111 increases α7-integrin in mdx muscle. Immunofluorescence reveals increased expression and extrajunctional localization of α7-integrin and utrophin in mdx mice. A further increase in α7-integrin expression was observed in mdx muscle treated with laminin-111. Rhodamine-labeled α-bungarotoxin was used to identify acetylcholine receptors at the neuromuscular junctions. (Scale bar: 10 μm.)

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
2.
Fig. 2.

Fig. 2. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Laminin-111 increases α7-integrin levels in mouse and human muscle cells. (A) Western blotting reveals increased levels of α7B-integrin in laminin-111-treated myoblasts compared with controls. Cox-1 was used as a loading control. (B) Quantitation shows a 2-fold increase in α7B-integrin in C2C12 myoblasts treated with laminin-111. *, P < 0.05. (C) Western blotting reveals increased α7B-integrin in laminin-111-treated DMD myoblasts compared with control. Cox-1 was used as a loading control. (D) Quantitation shows a 2-fold increase in α7B-integrin in DMD myoblasts treated with laminin-111.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
3.
Fig. 1.

Fig. 1. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Laminin-111 increases α7-integrin promoter activity in mouse muscle cells. (A) X-Gal staining demonstrating that α7βgal+/− myoblasts express β-galactosidase, which increases upon differentiation to myotubes. (Magnification: ×150) (B) Western blot analysis of α7βgal+/− myoblasts differentiated from 0–72 h shows a corresponding increase in both α7-integrin and β-galactosidase. α-Tubulin was used as a loading control. (C) FACS analysis reveals α7βgal+/− myoblasts exhibit increased β-galactosidase expression after 100 nM laminin-111 (LAM-111) treatment.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
4.
Fig. 5.

Fig. 5. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Laminin-111 promotes α7-integrin expression in mdx muscle. (A) Western blotting confirms the absence of dystrophin in mdx muscle treated with laminin-111 or PBS. A significant increase of α7-integrin was observed in TA muscle of mdx mice treated with laminin-111 compared with controls. (B) Quantitation reveals laminin-111-treated mdx muscle has a 1.6-fold and a 2.6-fold increase in α7A- and α7B-integrin, respectively, compared with control. A 1.3-fold increase in utrophin levels was observed in mdx muscle injected with laminin-111 compared with control. Protein loading was normalized to Cox-1. *, P < 0.05; **, P < 0.001; n = 5 mice per group.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
5.
Fig. 7.

Fig. 7. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Systemic laminin-111 protein therapy prevents exercise-induced muscle injury in mdx mice. (A) The mdx mice were treated with PBS or laminin-111 and 14 days later were subjected to a downhill running protocol and injected with EBD. Extensive EBD uptake in TA muscles of PBS-treated mdx mice indicates exercise-induced severe muscle damage. In contrast, laminin-111 protected mdx muscle from exercise-induced muscle injury. (Scale bar: 200 μm.) (B) EBD uptake was quantified in nonexercised mdx mice treated with PBS and exercised mdx mice treated with laminin-111 or PBS. Downhill running induced severe muscle damage in PBS-treated mdx mice. In contrast, laminin-111-treated mdx mice exhibited 28-fold fewer EBD-positive myofibers compared with PBS-treated animals. **, P < 0.001; n = 4 mice per group.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
6.
Fig. 3.

Fig. 3. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Intramuscular injection of laminin-111 prevents muscle disease in mdx mice. (A) Immunofluorescence of the TA muscles of control and laminin-111-treated mice confirms the absence of dystrophin in mdx muscle treated with LAM-111 or PBS. Laminin-111 was not present in wild-type or PBS-injected mdx muscle, but it was detected in the extracellular matrix of laminin-111-injected mdx muscle. (Scale bar: 10 μm.) (B) EBD uptake reveals that mdx muscle injected with laminin-111 exhibits reduced EBD uptake compared with control. (Scale bar: 10 μm.) H&E staining reveals that mdx muscle treated with laminin-111 contains few muscle fibers with centrally located nuclei and mononuclear cell infiltrate compared with control. (C) Quantitation reveals wild-type and mdx muscle treated with laminin-111 contained significantly fewer EBD-positive fibers and myofibers with centrally located nuclei compared with control. *, P < 0.05; **, P < 0.001; n = 5 mice per group.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.
7.
Fig. 6.

Fig. 6. From: Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

Systemic laminin-111 delivery prevents muscle disease in mdx mice. (A) Immunofluorescence reveals laminin-111 protein can be delivered systemically to cardiac, diaphragm, and gastrocnemius muscles. Endogenous laminin-111 was detected in treated and nontreated mdx kidney. The injected laminin-111 was detected in vena cava, blood vessels in the brain, and the liver. (Scale bar: 20 μm.) (B) RT-PCR was used to detect the laminin-α1 transcript in mdx skeletal muscle treated with PBS or laminin-111. A 260-bp laminin-α1 product was detected only in the kidney and not in the TA muscle of mice treated with PBS or laminin-111. 18S rRNA served as a control. (C) Quantitative TaqMan RT-PCR confirmed laminin-α1 transcript in kidney but not in the TA muscles of mice treated with PBS or laminin-111. 18S rRNA served as a control. (D) Serum creatine kinase was measured in wild-type or mdx mice 3 weeks after i.p. injections with PBS or laminin-111. Serum creatine kinase was elevated in PBS-injected mdx mice compared with wild type. In contrast, mdx mice injected with laminin-111 exhibit a 2.6-fold reduction in creatine kinase compared with PBS-treated mice and were not significantly different from wild type. *, P < 0.05; n = 5 mice per group. (E) Serum creatinine levels were unchanged between the groups. n = 5 mice per group. (F) Blood urea nitrogen levels were unchanged between the groups. n = 5 mice per group.

Jachinta E. Rooney, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7991-7996.

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