AMPK Regulates HSP70 Figure 4. 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 42uC 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 6 SEM, n = 3. Representative gels were shown. Nuclear pore complex protein gp62 (GP62) was shown for protein loading. D. HepG2 cells were exposed to 42uC for 1 h, then treated with 5 mg/ml actinomycin D in the presence or absence of 1 mM AICAR and kept at 42uC 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 6 SEM, n = 3. B–C: experiments were performed at least three times and the representative results are shown. doi:10.1371/journal.pone.0013096.g004 HSP70 expression at least partially through the increase of HSP70 mRNA stability. Metabolic stress induced AMPK activation can inhibit protein synthesis through two pathways: inhibition of the elongation step of translation through activation of elongation factor-2 kinase, and inhibition of the initiation step for protein synthesis through inhibiting target-of-rapamycin (TOR) pathway [1]. It’s possible that the inactivation of AMPK could enhance protein synthesis. Inactivation of AMPK under heat stress may contribute to HSP70 protein synthesis but needs further investigation. Besides the metabolic activity of AMPK, there is growing evidence that AMPK activation plays negative roles in cell survival and growth [3,4,23]. HSPs are critical components of defense mechanism against injury associated with adverse stresses [24]. Our results for the first time demonstrated the involvement of AMPK in modulation of heat shock protein gene expression which is a novel mechanism of heat shock response. Inhibition of AMPK under heat stress and the link between AMPK and HSP may be beneficial to cell survival. Materials and Methods Cell culture and treatment All cells were cultured in DMEM containing 10% FBS and antibiotics. Short-time heat stress (#1 h) was performed by submersion of cells in a pre-warmed circulating water bath at 42uC. For long-time heat shock stress, the cells were incubated at 42uC in a humidified 5% CO2 atmosphere incubator. AICAR was purchased from Cell Signaling Technology (New England Biolabs, Beverly, MA). CdCl2, sodium azide (NaN3) and 2-Deoxyglucose were from Sigma (Louis, MO). Celastrol, MG132 and actinomy- cin D were from Calbiochem (Darmstadt, Germany). siRNA transfection bEnd.3 or HepG2 [25] cells were transfected with siRNA by using lipofectamine 2000 (Invitrogen). The final concentration of siRNA was 30 nM. AMPKa1/2 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA). The duplexes of siRNA targeting PP2A catalytic-subunit mRNA (targeting sequence 59-GAATC- CAACGTTCAAGAGG-39) and control siRNA (scrambled se- quence) were synthesized by GenePharma (Shanghai, China). ATP assay The intracellular ATP contents were detected by ENLITENH ATP Assay System Bioluminescence Detection Kit (Promega) according to manufacturer’s instructions. Real-time PCR Total RNA was extracted from cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and depleted of contaminating DNA with RNase-free DNase. cDNA was synthesized from 2 mg RNA with M-MuLV reverse transcriptase and random hexamer (Fermentas, Burlington, Ontario, Canada). Reverse-transcribed cDNA in triplicate samples were checked for target mRNA level by quantitative real-time PCR with Power SYBR Green PCR master Mix (Applied Biosystems Inc, Warrington WAI4SR, UK) PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13096