Adenosine

To examine the effect of adenosine on ΔΨ_m in HSCs in vitro, HSCs were cultured under undivided conditions (0.5 ng/ml SCF and 0.5 ng/ml TPO) with 0.1∼10 µM adenosine (Sigma) for 48 h and subjected to JC-1 staining. To investigate the effect on intracellular Ca²⁺ level, immediately after Fluo-4–labeled HSCs were stimulated with 1 µM adenosine, the fluorescent intensity of Fluo-4 was examined by flow cytometry. To examine the role of adenosine A2 receptors in ΔΨ_m or intracellular Ca²⁺ level under divided conditions (50 ng/ml SCF and 50 ng/ml TPO), HSCs were treated with 50 µM CV1808 (R&D Systems) for 48 h and subjected to JC-1 or Fluo-4 staining. For in vivo study, 3 mg/kg CV1808 were administrated via i.v. two or three times every 24 h from 1 d after 5-FU treatment, and subsequently ΔΨ_m or EdC uptake in HSCs were examined at 3 or 4 d after 5-FU administration, respectively. Moreover, both 6 mg/kg SCH442416 and 6 mg/kg PSB1115 were administrated via i.p. four times every 24 h from 3 d after 5-FU treatment. These treated mice were analyzed at 7 d after 5-FU administration.