ggest that TSA treatment induces the movement of HIF-1α into the nucleus for binding to the HRE of the VEGF promoter.

![TSA treatment increased HIF-1α binding on HRE of VEGF promoter\
HeLa cells were transfected with pSG5-HIF-1α, treated with TSA and fixed with 10% formaldehyde. Their chromatin extracts were immunoprecipitated with anti-GST antibodies. DNA fragments were subjected to PCR analysis using primer sets spanning HIF-1α binding site (--1,037 \~ --869 bp) of VEGF gene promoter regions (**A**). Cell lysates were prepared and immunoprecipitated with anti-GST antibodies. HIF-1α was detected with western blot analysis (**B**, left). Each band was quantified by using IamgeJ 1.34 and the results were represented as fold changes to control. (B, right). VEGF-HRE activity was measured by using luminometer. Data are the representative of three experiments. Data in bar graph represent mean ± SD. ^\*\*^*p* \< 0.01, significantly different from TSA-untreated control group transfected with mock or pSG5-HIF-1α. ^&&^*p* \< 0.01, significantly different from pSG5 control vector-transfectd group in the absence of TSA. ^\#\#^*p* \< 0.01, significantly different from pSG5 control vector-transfectd group in the presence of TSA (**C**).](oncotarget-09-2035-g004){#F4}

TSA treatment increases the acetylation of HIF-1α at lysine 674 {#s2_5}
---------------------------------------------------------------

Because TSA is an HDAC inhibitor \[[@R13]\], we determined whether the acetylated lysine (K) is the mechanism underlying the nuclear localization of HIF-1α induced by TSA. When cells were transfected with pSG5-HIF-1α and treated with TSA, acetylation was increased in many proteins (Figure [5A](#F5){ref-type="fig"}). HIF-1α acetylation was detected by immunoprecipitating GST-HIF-1α and immunoblotting acetylated lysine (Figure [5B](#F5){ref-type="fig"}). We also determined whether endogenous HIF-1α might be localized into nucleus via lysine acetylation induced by TSA. As shown in Figure [5C](#F5){ref-type="fig"}, ac