AMPK was activated in ECFC differentiation via KLF2 up-regulation. (A and B) Human ECFCs were treated with AICAR for various times. KLF2 mRNA level was examined by Q-PCR after 12 h of treatment (A). Western blot analysis of protein levels of AMPK, eNOS, and KLF2 after 1 or 24 h of treatment (B). (C and D) ECFCs were pre-treated with dimethyl sulfoxide or Compound C (10 μM) for 30 min, then further incubated with or without VEGF (25 ng/mL) for 24 h. Q-PCR analysis of mRNA levels of KLF2, eNOS, ICAM-1, CD31, CD34, and c-kit (C). Western blot analysis of protein levels of KLF2, eNOS, and phosphorylated eNOS (D). β-actin was an internal control. Data are mean ± SD from three independent experiments, each performed in triplicate (*P < 0.05; **P < 0.01).