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. 2010 May 17;120(6):2016–2029. doi: 10.1172/JCI39896

Figure 7. EPO upregulates expression levels of Shh.

Figure 7

Western blotting of Shh in cultured neonatal rat cardiomyocytes and cardiac fibroblasts (A) and in the culture supernatants (B). Cells were treated with EPO (100 U/ml) or CEPO (100 U/ml) for 48 hours. Shh-N represents the aminoterminal domain of Shh, which is a biologically active form of Shh. (C) Immunocytochemical staining for Shh (red), α-sarcomeric actinin (green), and vimentin (blue). Cardiomyocytes and cardiac fibroblasts were cocultured with or without EPO for 48 hours. EPO induced the cytoplasmic accumulation of Shh in cardiomyocytes but not cardiac fibroblasts. Scale bars: 10 μm. (D) qRT-PCR analysis of Shh mRNA. Cardiomyocytes were treated with EPO for 24 hours (n = 5). *P < 0.05. (E) qRT-PCR analysis of Ptch-1, Gli-1, and Ang-1 mRNA. Cardiomyocytes were treated with rmShh (0.1 or 1.0 μg/ml) for 24 hours (n = 4). *P < 0.05; **P < 0.01 versus control. #P < 0.05; ##P < 0.01 versus rmShh and cyclopamine (5 μM) treatment. (F) Western blotting of VEGF. Cardiomyocytes were treated with rmShh (1.0 μg/ml) for 48 hours. Cyclopamine (cyclo) was administered 15 minutes before rmShh treatment. Representative Western blots and quantification of the bands are shown (n = 4).