RNF5 in ER-Related Disorders Figure 4. RNF5 induced myodegeneration is associated with extensive myofiber regeneration. A. Modified Gomori-trichrome staining of triceps brachii cross-sections from treated STg and DTg animals. DTg muscle sections show a number of degenerative fibers with cellular infiltration (arrows). B. Serum creatine kinase (CK) levels were monitored in RNF5 STg and rtTA RNF5 DTg after 4 wk of doxycyclin treatment (n = 3). DTg animals had variably elevated CK levels. C. Dye-permeable fibers are surrounded or invaded by macrophagic inflammatory cells clearing necrotic debris. Treated DTg and control mice were injected with Evans Blue Dye (EBD) 12 hr before sacrificed and fiber permeability to the dye was monitored externally by the blue coloration of the muscles (right panel) and after cross-section (red fluorescence; left panel). The muscle cross-sections were counterstained with an antibody against dystrophin to visualize fiber boundaries and to highlight degenerative myofibers where staining for dystrophin was absent. D. Ultrastructural analysis of DTg muscle sections. In longitudinal sections, sarcomere structure was normal in non- degenerating myofibers (left, asterisks). In an adjacent degenerating fiber, disruption of sarcomere structure, dilated tubular structures (arrows), and enlarged mitochondria (arrowheads) are evident. A similar pattern was evident in cross-section (right panel). Magnification611,000. E. Muscle cross- sections of doxycyclin-treated DTg and control animals stained with antibodies against embryonic myosin heavy chain (embMHC) and myogenin. Arrowheads indicate some nuclei exhibiting a positive myogenin staining in sections from DTg animals. Muscle sections were counterstained with anti-laminin a3 antibody (red) and DAPI (blue) to visualize respectively myofiber boundaries and nuclei. F. Quantification of the number of regenerative myofibers, expressed as the average number of embMHC positive fibers (early regenerative fibers) and of centrally nucleated fibers. G. Mice overexpressing RNF5 have extensive mononuclear cell infiltration in areas of myofiber degeneration. Cd11b immunostaining (arrowheads) highlight macrophagic infiltration of triceps brachii muscle cross-sections from rtTA RNF5 DTg (b) but not RNF5 STg (a) animals treated with doxycyclin for 6 wk. Nuclei are stained with DAPI (blue). Negative control (mouse IgG) performed on sections from rtTA RNF5 DTg is shown in panel (c). (d) Muscle regeneration associated with RNF5 overexpression does not result in fibrosis. Triceps cross-sections from RNF5 STg and DTg animals were stained with modified Gomori-Trichrome. doi:10.1371/journal.pone.0001609.g004 PLoS ONE | www.plosone.org 7 February 2008 | Volume 3 | Issue 2 | e1609