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Fig. 7. Zfp64 inhibits myogenesis and promotes the expression of osteogenic marker genes. (A) Zfp64 and NICD inhibit the expression of muscle-specific cytoskeletal proteins. Tet-on-NICD-C2C12 cells were treated as described in Results and western blot analysis was performed. The band specific for desmin is shown by the arrow and the nonspecific band is indicated by an asterisk. (B) siRNA-mediated gene knockdown efficiency. Left. C2C12 cells were co-transfected with AdZfp64 and AdsiZfp64 in the indicated ratios. Western blot analysis was performed 1 day after transfection. (Right) C2C12 cells were transfected with mock or AdsiZfp64. Real-time PCR was performed 3 days after transfection. (C) Zfp64 and NICD inhibit the expression of myogenic transcription factors. Tet-on-NICD-C2C12 cells were treated as described in Results and real-time RT-PCR was performed. Data shown are representative of two independently conducted experiments (mean ± s.e.). (D) Myotube formation four days after myogenic induction, visualized by immunofluorescent staining using antibody against sarcomeric actin. (E) Alkaline phosphatase (ALP) activity of C2C12 cells increased by Zfp64. C2C12 cells were transfected with AdGFP, AdZfp64 or AdsiZfp64. Twelve hours after transfection, cells were treated with 20 ng/ml human recombinant BMP2 protein for 3 days and ALP activity was evaluated. (Top) ALP staining of cells. (Bottom) Relative ALP activity measured using pNPP substrate. Data are expressed as the mean ± s.e. of triplicates. (F) C2C12 cells were treated as described in E, then the expression of type I collagen (Col1a1), osteocalcin (Ocn) and osteopontin (Opn) was examined by real-time RT-PCR. Multiple independently conducted experiments were performed and representative data are shown as the mean ± s.e. of triplicates. (G) Relative ALP activity of HDC cells transfected with AdGFP, AdZfp64 or AdHey1. Data are expressed as the mean ± s.e. of triplicates. (H) Relative expression level of Col1a1, Ocn and Opn of HDC cells transfected with AdGFP, AdZfp64 or AdsiZfp64 (mean ± s.e.).
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