Effects of AMPK activation on HSF1 activity and HSP70 mRNA stability under heat stress.
A–C. HepG2 cells pretreated with or without different concentrations of AICAR for 15 min were exposed to 42°C for 30 min, then examined for nuclear HSF1 protein level (A) and HSF1 phosphorylation (B) by Western blot, or HSF1/HSE binding by EMSA (C). A: mean ± SEM, n = 3. Representative gels were shown. Nuclear pore complex protein gp62 (GP62) was shown for protein loading. D. HepG2 cells were exposed to 42°C for 1 h, then treated with 5 µg/ml actinomycin D in the presence or absence of 1 mM AICAR and kept at 42°C for indicated time intervals. HSP70 mRNA levels were examined by real-time PCR. Exponential decay curves of HSP70 mRNA were plotted to calculate the half life of HSP70 mRNA. Mean ± SEM, n = 3. B–C: experiments were performed at least three times and the representative results are shown.