HSF1-Dependent COX-2 Induction by Heat Figure 5. In vitro and in vivo binding of HSF1 to the COX-2 promoter. (A) Nucleotide sequence of the 100 bp (from 22579 to 22480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32P-labeled and used as probe for gel-shift analysis shown in B and C. (B) HUVECs were subjected to heat shock at 43uC or left untreated (control). After 20 and 40 min at 43uC (HS) or at the indicated times during recovery at 37uC (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in (A). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and b-actin proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. (C) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43uC for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B. (D,E) HUVECs were subjected to heat shock at 43uC or left untreated (control). After 20 and 40 min at 43uC (HS) or at the indicated times during recovery at 37uC (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. (D) ChIP-enriched DNAs using preimmune serum (IP NS IgG) or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (22629 to 22420) and HSP70 gene (2262 to 270) were amplified by PCR. (E) Quantification of ChIP assay shown in (D). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate 6 S.D. * = P,0.05. doi:10.1371/journal.pone.0031304.g005 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31304