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Fig. 8. Knock-down of MURF-2 levels in chick skeletal myoblasts perturbs 
-tubulin and desmin staining and significantly inhibits fusion. (A) Skeletal myoblasts were treated with antisense or control oligonucleotides and fixed at 24 or 48 hours. The cells were stained with antibodies to -tubulin (b,f,j,n) and with DAPI (c,g,k,o) to quantify the number of nuclei/myotube as a measure of fusion. Within 24 hours of treatment, thick control myotubes contained diffuse MURF-2 staining, bright filamentous microtubules parallel to the longitudinal axis, and bright, diffuse desmin staining (a,b,d). Control myotubes also had a mean of 
16 nuclei (c,B) (note: the myotube in c also had nuclei outside the field of view). In contrast, antisense-treated myotubes were strikingly thin, had low levels of MURF-2 staining (e), low -tubulin and desmin staining (f,h) and significantly fewer nuclei (g,B). Within 48 hours of treatment, MURF-2 levels were comparable in antisense-treated myotubes (m) and control myotubes (i), and -tubulin staining in both populations of myotubes appeared in bright filaments (j,n). Desmin staining appeared bright and diffuse in both populations of myotubes (l,p). However, the MURF-2 antisense treated myotubes remained thinner and qualitatively contained fewer nuclei/myotube compared to controls (k,o). Note that the large number of overlapping nuclei/myotube could not be quantified accurately at 48 hours in control myotubes. Bar, 10 µm. (C) Human GFP-MURF-2 expression rescues fusion events in antisense-treated chick skeletal myotubes. Chick skeletal myoblasts were co-transfected with MURF-2 antisense oligos and human GFP-MURF-2, fixed 24 hours after treatment, and stained with DAPI to quantify the number of nuclei per myotube. Myotubes expressing GFP-hMURF-2 (n=15 from two different cultures) contained similar numbers of nuclei/myotube compared to myotubes treated with control oligonucleotides; both populations contained significantly greater numbers of nuclei/myotube compared with antisense-treated myotubes not expressing human MURF-2-GFP.
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