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Fig. 7. Cyr61, an NFAT5 target gene in skeletal muscle, regulates myoblast motility. (A) RNA was isolated from primary myoblasts (Mb), nascent myotubes (nMt) and mature myotubes (mMt) as well as from murine kidney (+). RT-PCR analyses demonstrated that Cyr61 was expressed in skeletal muscle cells at all stages of myogenesis. Equivalent input of cDNA was confirmed by amplification of the ribosomal gene 18S. (B) Immunoblot analyses demonstrated that Cyr61 protein was expressed in both uninjured (C) and regenerating (n=3 animals at each time point except n=2 for day 5) TA muscles at days 1 and 5 after injury. Equivalent protein loading was confirmed by Coomassie Blue staining. (C) Lysates from myoblasts containing either control (C) or dominant-negative NFAT5 (DN) plasmids were immunoblotted for Cyr61. Cyr61 was decreased in DN cells. Tubulin was detected as a loading control. (D) The average velocity of myoblasts expressing DN was decreased relative to the control. (*P<0.05). Average velocity returned to control levels following the addition of 5 µg/ml Cyr61 to these cells (**P<0.05 relative to DN). Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates). (E) Frequency histogram illustrating the distribution of cell velocities for control, DN and DN+Cyr61 cells. The DN curve was shifted to the left relative to control. The addition of 5 µg/ml Cyr61 to DN cells caused a rightward shift in the population. Data are mean ± s.e.m. of 60 cells (20 cells from each of three independent isolates).
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