A significant reduction of reporter activity was observed www.selleckchem.com/products/ABT-263.html with 50 ��M and 100 ��M sirtinol (Figure 2A). We next verified that inhibition of SIRT1 activity alters the transcriptional activity of HIF-1 target genes. Pretreatment with 100 ��M sirtinol significantly reduced the hypoxic induction of specific HIF-1 targets BNIP3 and CA9 mRNA as well as EPO mRNA (Figure 2B). Although, sirtinol specifically inhibits SIRT1, it also affects other members of the sirtuin family such as SIRT2 with an IC50 value of 40 ��M [41]. Therefore to verify that the effect of sirtinol on repressing HIF-mediated transcriptional activity is at least in part due to the inhibition of SIRT1, cells were infected with lentiviruses carrying shRNA sequences targeting SIRT1.

Targeted disruption of SIRT1 with clone shSIRT1_1958 led to a nearly complete knockdown whereas, shSIRT1_3206 resulted in a partial knockdown of SIRT1 protein compared to parental and SHC002 controls (Figure 2E). Cells infected with lentiviruses expressing clone shSIRT1_1958 significantly reduced the hypoxic induction of CA9 and EPO mRNA (Figure 2C). These data demonstrate that inhibition of SIRT1 activity with a small molecule inhibitor and a genetic knockdown leads to a strong decrease of HIF-1-mediated transcriptional activity. Figure 2 SIRT1 inhibition represses HIF-1 transcriptional activity and HIF-1�� protein. We next questioned, whether loss of HIF transcriptional activity by inhibiting SIRT1 was due to an effect on HIF-1�� protein itself. Hep3B cells were incubated with sirtinol for 16 hours and then exposed to 1% O2 for 4 hours.

HIF is mainly regulated by hydroxylation of its ��-subunit, so as expected, in normoxic Hep3B cells HIF-1�� protein was degraded and nearly undetectable, whereas it was efficiently stabilized under hypoxia. Interestingly, inhibition of SIRT1 activity with sirtinol led to a dose-dependent repression of HIF-1�� protein accumulation (Figure 1D). Importantly, HIF-1�� was detected by using an antibody that is specific for HIF-1�� and does not cross react with HIF-2�� [43]. To verify that the repressive effect on HIF-1�� expression was due to SIRT1 inhibition, the experiment was repeated in Hep3B with a knockdown of SIRT1 expression. SIRT1 knockdown cells had an impaired ability to accumulate HIF-1�� protein; the efficiency of SIRT1 knockdown correlated with the suppression of HIF-1�� protein, with a stronger effect achieved in cells infected with lentivirus shSIRT1_1958 (Figure 2E).

To determine the kinetics of sirtinol-mediated HIF-1�� protein repression, Hep3B cells were either incubated with sirtinol for 16, 2 and 0 hours before exposing them to hypoxia for 4 hours or sirtinol was added to the cells 2 hours after exposure to hypoxia. Sirtinol added at the onset of hypoxia inhibited the accumulation of HIF-1�� protein. An even stronger repressive effect Drug_discovery was observed when cells were pre-exposed to sirtinol for 2 or 16 hours.