Stabilization of HIF-1�� protein led to sellectchem the transcriptional increase of HIF-1 target genes BNIP3, CA9 as well as EPO, a target gene shared with HIF-2 (Figure 1B). To further verify that SIRT1 protein is not altered by hypoxia, HCC cells were exposed to 1% O2 for up to 48 hours. There was no change in SIRT1 protein or mRNA in cells exposed for 12, 24 or 48 hours to 1% O2 (Figure 1C & D). In addition, there was no significant change in SIRT1 mRNA in Hep3B cells exposed to more severe hypoxia of 0.1% O2 for 24 hours (Figure 1E). These data show that both SIRT1 and HIF-1�� are present in cells under hypoxic conditions and HIF-1 is transcriptionally active. Moreover, unlike HIF-1��, SIRT1 expression is not tightly regulated by hypoxia in HCC cells. Figure 1 SIRT1 and HIF-1�� are simultaneously expressed in hypoxic cells.

Inhibition of SIRT1 decreases HIF transcriptional activity and suppresses HIF-1�� protein accumulation As demonstrated above, we observed a simultaneous expression of both SIRT1 and HIF-1�� protein under hypoxic conditions. A recent report by Lim et al. has suggested that SIRT1 negatively regulates HIF-1 activity by suppressing the transcriptional regulation of its target genes [27]. Following the model proposed by their data, inhibition of SIRT1 should provoke an exaggerated HIF-mediated response. Therefore, we tested the effect of inhibiting SIRT1 on HIF-1 activity using sirtinol, a cell permeable specific inhibitor of SIRT deacetylase activity [41]. The IC50 value of sirtinol is 70 ��M for SIRT1 activity; hence concentrations of 25, 50 and 100 ��M were used in this study.

First, we confirmed the functionality of sirtinol on SIRT1 deacetylase activity in HepG2 cells. HepG2 cells have abundant SIRT1 protein, as well as an intact p53 response to DNA damage. The acetylation of lysine 382 of p53 is modulated by SIRT1 [21]. We demonstrated that sirtinol had no detectable effect on the lysine 382 acetylation in the absence of DNA damage, whereas, sirtinol produced a time-dependent increase of acetylated p53 in cells treated with the DNA damaging agent, doxorubicin (Figure S1). These data confirm that sirtinol is an efficient inhibitor of SIRT1 activity in vitro. We next investigated the effect of inhibiting SIRT1 activity on HIF-mediated transcriptional activation.

Cells were transfected with a HRE reporter construct that contains multiple HRE sites and is specifically induced by HIF proteins and thus represents a direct measurement Carfilzomib of HIF activation [42]. Twenty-four hours after transfection, cells were treated with increasing doses of sirtinol and incubated 24 hours at 1% O2. Hypoxia increased reporter activity 38-fold and there was no significant change in cells treated with DMSO at a concentration equal to the amount needed to treat cells with 100 ��M sirtinol.