The secondary antibody was FITC-conjugated anti-rat antibody at a concentration of 1:200. The alkaline phosphatase activity was also assessed using the alkaline phosphatase kit (Sigma). The staining procedure was done according to

the manufacturer’s instruction. Results Cytotoxicity Assay The cardiomyocyte extract was not toxic; consequently, 92.3% of the unpermeabilized cells exposed to the extract for one h were viable. The extract-treated cells as well as the control cells that were Inhibitors,research,lifescience,medical treated with only HBSS were able to grow after extract exposure. Neutral red staining revealed that the cells were viable after culturing for 24 h. Permeabilization Assay FITC-dextran uptake was observed in the cells that were exposed to this marker in the presence of Inhibitors,research,lifescience,medical 230 ng/mL of streptolysin O. The cells treated with FITC-dextran without permeabilization with streptolysin O were also able to uptake the marker; however, the fluorescence intensity and the number of the cells that showed fluorescence were less Inhibitors,research,lifescience,medical than those in the streptolysin O-treated cells. This

may be related to endocytosis, which took place during the incubation, as has been reported by other researchers.13 The cells were allowed to culture for 24 h. The cells were able to expand and survive, as was indicated by the neutral red assay. Cell Morphology The administration of both 5-aza-dC and TSA reduced cell growth, as was indicated by the number of the passages. Also, 5-aza-dC, when treated alone, had no influence on cell growth. Extensive cell death was Inhibitors,research,lifescience,medical observed with TSA exposure. Although the viable cells were able to proliferate, the confluency was not more than 50%. These chromatin-modifying agents also changed cell morphology. The treated cells were larger than those cultured in the absence of chromatin-modifying agents. The number of processes was reduced, and the cells were polyhedral in shape. More conspicuous morphological modification was observed in the other cell types such as human Inhibitors,research,lifescience,medical granulosa cells and mouse fibroblast cell line (NIH3T3): they became fusiform as a result

of treatment with the chromatin-modifying Carnitine palmitoyltransferase II agents. (Data are not shown.) After extract treatment, more cells showed morphological changes. The cells became elongated and lost their processes. Some multinucleated cells with two or three nuclei were also observed (figure 1). There was no Selleckchem LY2157299 beating cell in the culture with this condition. The cell proliferation rate reduced significantly; however, the cells were viable for at least 30 days. While the cells in the control groups needed to passage every 3 days, the extract treated cells were not confluent even after 30 days from the beginning of the exposure to the cardiomyocyte extract. Figure 1 Cells treated with the extract and chromatin-modifying agents were multinucleated.