PP2A, and found that OA efficiently reversed heat stress-induced dephosphorylation of AMPKa (Figure 1F). As a positive control, AICAR also reversed heat stress –induced AMPKa dephosphor- ylation. Okadaic acid can also inhibit PP1, but it inhibits PP1 activity with higher concentration [16]. 100 nM okadaic acid has been reported to inhibit PP2A but not PP1 [16,17]. Therefore, our results suggest that PP2A may inhibit AMPK upon heat stress. To further confirm that heat stress inhibits AMPK through activation of PP2A, we inhibited PP2Ac expression by RNA interference (Figure 1G). Transfection with PP2Ac siRNA but not control siRNA restored the phosphorylation level of AMPK after the cells were exposed to heat stress (Figure 1H), supporting that heat stress-induced AMPK inhibition is due to the activation of PP2Ac. AMPK inhibition under stress contributes to HSP70 upregulation The dramatic induction of heat shock proteins is an unifying and adaptive response that enhances cell survival from stress. HSP70 is one of the most highly induced heat shock proteins in response to heat stress. So we examined the relationship between AMPK inhibition and HSP70 upregulation under heat stress. As shown in Figure 2A and B, heat stress significantly induced HSP70 expression at both mRNA and protein levels in HepG2 cells. AMPK Regulates HSP70 Pretreatment of the cells with AICAR significantly inhibited heat- induced HSP70 expression. Similar results were obtained from nonspecific siRNA transfected HepG2 cells (Figure 2D and E). When HepG2 cells were transfected with AMPKa1/2 specific siRNA to inhibit AMPKa expression (Figure 2C), the inhibitory effect of AICAR on heat stress-induced HSP70 expression was partially reversed at mRNA level and totally reversed at protein level (Figure 2D and E). These results demonstrated that activation of AMPK by AICAR significantly inhibited HSP70 expression in response to heat stress, which suggests that inhibition of AMPK under heat stress contributes to HSP70 upregulation. AMPK, as a cellular energy sensor, is activated by the reduction of cellular ATP levels. To get more evidence for negative regulation of HSP70 expression by AMPK under heat stress, we used NaN3 combined with 2-DG (2-deoxyglucose) to activate AMPK. Treatment of HepG2 cells with these two compounds significantly induced AMPK phosphorylation (Figure 2F) and inhibited heat stress-induced HSP70 expression at both mRNA and protein levels (Figure 2G and H). These results further support that HSP70 is negatively regulated by AMPK under heat stress. We also checked the involvement of AMPK in HSP70 upregulation in response to other stressors which could induce HSP expression. As shown in Figure 3A and B, stimulation of HepG2 cells with celastrol, CdCl2, or MG132, significantly Figure 2. Activation of AMPK inhibits HSP70 expression in response to heat stress. A–B. HepG2 cells pretreated with or without 1 mM AICAR for 15 min were exposed to 42uC (HS) for 1 h, HSP70 expression was examined by real-time PCR (A) and Western blot after recovery at 37uC for 5 h (B). mean 6 SEM, n = 3. **P,0.01 vs. cells without heat stress; ##P,0.01 vs. cells cultured in control medium under heat stress. C–E. HepG2 cells transfected with AMPKa siRNA or control siRNA for 48 h were examined for AMPKa protein level by Western blot (C), or treated with or without 1 mM AICAR for 15 min followed by exposure to 42uC for 1 h, then examined HSP70 expression by real-time PCR (D) and Western blot after recovery at 37uC for 5 h (E). mean 6 SEM, n = 3. **P,0.01 vs. control siRNA transfected cells without AICAR treatment under heat stress; #P,0.05, vs. AMPKa siRNA transfected cells without AICAR treatment under heat stress. F. HepG2 cells were treated with 2 mM NaN3 combined with 50 mM 2- Deoxyglucose (NDG) for 30 min, then examined for AMPKa phosphorylation by Western blot. G–H. HepG2 cells pretreated with or without NDG for 30 min were exposed to 42uC for 1 h, HSP70 mRNA was examined by real-time PCR (G), HSP70 protein level was examined by Western blot after recovery at 37uC for 5 h (H). mean 6 SEM, n = 3. **P,0.01 vs. cells without heat stress. ##P,0.01 vs. cells without NDG treatment under heat stress. B, C, E, F and H: experiments were performed at least three times and the representative results are shown. doi:10.1371/journal.pone.0013096.g002 PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13096