Figure 3

AMPK activation by metformin increases the biogenesis of let-7, which requires KSRP. (a, b) ARK2 cells were treated with AICAR (final concentration 0.5 mm), followed by RNA extraction and reverse transcriptase (RT)–quantitative PCR (qPCR) to determine RNA levels of let-7 (a) and H19 (b). (c) ARK2 cells were treated with or without metformin or AICAR, followed by western blot analysis for phosphorylated AMPK (p-AMPK) and total AMPK (AMPK) using β-actin (ACTB) as a loading control. Representative western blot gels of three independent experiments are shown. (d, e) ARK2 cells were transfected with siCon or Ksrp-specific siRNA (siKsrp), followed by RT–qPCR (d) and western blot analysis (e) to confirm KSRP knockdown at the mRNA and protein levels, respectively. (f, g) ARK2 cells were transfected with siCon or siKsrp, followed by treatment with or without metformin 24 h later. RNA levels of let-7 (f) and H19 (g) were determined by RT–qPCR 24 h post-transfection. Numbers are mean±s.d. (n=3). **P<0.01. *P<0.05. NS, no statistical difference.