RNF5 in ER-Related Disorders Figure 5. RNF5 localizes to the ER and its overexpression correlates with altered ER function. A. Expression levels of select ER stress markers in muscles of DTg animals are elevated compared to their control littermates. Western blot analysis of muscle extract (80 mg) from RNF5 STg or rtTA RNF5 DTg animals treated for 6 weeks with doxycyclin was probed with Grp78, PDI, Grp94, calnexin and CHOP antibodies. RNF5 and GAPDH antibodies were used for RNF5 expression and loading controls, respectively. B. Grp94 immunostaining of vastus lateralis cross-sections from treated rtTA RNF5 DTg and STg animals. C. RNF5 localizes to the ER of muscle fibers. Vastus lateralis cross-sections from treated DTg animals were stained with RNF5 antibody. Normal (top panels) or regenerative fibers (bottom panels) are shown for RNF5 staining alone (left panels) or combined with DAPI (right panels). doi:10.1371/journal.pone.0001609.g005 Long-term muscle-specific overexpression of RNF5 results in muscle degeneration and regeneration and vacuole formation In order to analyze the effect of RNF5 overexpression in a tissue specific manner and to confirm that the phenotype observed originates from RNF5 expression in mature muscles, we crossed RNF5 transgenic animals with mice expressing the tTA transgene under the control of the muscle specific promoter MCK (Muscle Creatine Kinase) (Fig. 6A). This mouse model was previously generated to control conditional gene expression in skeletal muscles in a tetracyclin repressible manner [32]. In this model, double transgenic animals (MCK-tTA RNF5 DTg) and their matching controls (RNF5 STg) were kept under doxycyclin treatment until they reached 3 months of age to prevent the induction of the RNF5 transgene. Specific overexpression of RNF5 in skeletal muscle was then induced following doxycylin with- drawal. RNF5 level of expression was comparable to the one we observed using b-actin promoter (Fig. 6B). When expressed from a muscle-specific promoter, RNF5 overexpression led to a less severe, albeit clear and reproducible clinical phenotype, with animals surviving up to 5 month after initiating transgene induction. MCK-tTA RNF5 DTg were found to exhibit a mild weight loss over time, compared to STg (Fig. 6C). This milder phenotype enabled us to monitor the effect of RNF5 overexpression over a prolonged time period. While MCK-tTA RNF5 DTg animals exhibited histopathological changes similar to those observed in the b-actin expressing RNF5 mice, only few animals exhibited these phenotype within the same time frame seen for rtTA RNF5 DTg mice (6 wk). The majority of MCK-tTA RNF5 DTg mice exhibit a clear muscle phenotype only 20 wk after doxycyclin withdrawal (Fig. 6D panels a,b). Strikingly, in addition to the classical myodegeneration and regeneration process previously observed in the rtTA RNF5 DTg model, prolonged expression of RNF5 in the MCK-tTA RNF5 DTg also caused the appearance of Congo Red positive fibers (Fig. 6D PLoS ONE | www.plosone.org 8 February 2008 | Volume 3 | Issue 2 | e1609