Fig. 3.

Nampt is released from cardiomyocytes under stress. A and B: primary cultures of cardiomyocytes were infected with 10 or 100 multiplicity of infection (MOI) of adenovirus expressing Nampt (Ad.Nampt) for 24 h. Cont, control. Supernatant was analyzed for the presence of Nampt by ELISA, and cells were analyzed for the Nampt expression levels by immunoblotting. C: equal amount of supernatants from different group of cells were analyzed for the presence of Nampt by immunoblotting with anti-Nampt antibody. D and E: primary cultures of cardiomyocytes were serum starved for 24 or 48 h, the supernatants were analyzed for the presence of Nampt by ELISA, and cells were analyzed for the Nampt expression levels by immunoblotting. F and G: primary cultures of cardiomyocytes, serum starved for 6 h, were treated with nonlethal dose of H₂O₂ (50 μM) for 1, 2, and 4 h. Supernatants were analyzed for the presence of Nampt by ELISA, and cells were analyzed for the Nampt expression levels by immunoblotting. H and I: primary cultures of cardiomyocytes were serum starved for 24 h and then treated with 25 mM nicotinamide (NAM) or 500 nM trichostatin A (TSA). After 24 h of treatment, supernatants were analyzed for the presence of Nampt by ELISA, and cells were analyzed for the Nampt expression levels by immunoblotting. Means ± SD; n = 4–6.