HSF1-Dependent COX-2 Induction by Heat Figure 2. Kinetics of heat-induced COX-2 expression and PGE production in endothelial cells. A–C. HUVECs were either kept at 37uC (Control) or were subjected to heat shock (HS) at 43uC for 40 min. At the end of heat treatment, or at 1.5, 3, 6 and 8 hours during the recovery period at 37uC, total RNA was analyzed for COX-2, COX-1, HSP70 and b-actin mRNA by RT-PCR (A) and real-time PCR (B). In parallel samples whole-cell extracts were analyzed for levels of COX-2, HSP70 and b-actin proteins by Western blot (C). (D) Immunofluorescence of HUVECs kept at 37uC (Control) or subjected to heat shock at 43uC for 40 min and allowed to recover at 37uC for 3 hours (HS) labeled with anti-COX-2 antibodies (red). Nuclei are stained with DAPI (blue). The overlay of the two fluorochromes is shown (Merge). Images were captured with a Leica confocal microscope TCS 4D system. (E) HUVECs were either kept at 37uC (Control) or were subjected to heat shock (HS) at 43uC for 40 min. At 2, 4 and 6 hours during the recovery period at 37uC, whole-cell extracts were analyzed for levels of COX-2 and b-actin proteins by Western blot (top panel), and PGE2 production in the culture supernatants was determined by ELISA (bottom panel). In parallel samples treated with IL-1b (20 ng/ml) COX-2 levels and PGE2 production were determined 6 hours after treatment. (F) HUVECs were treated with the COX-2 inhibitor NS398 (100 mM) or DMSO vehicle either 1 hour before or during HS at 43uC for 40 min. At 2 hours during the recovery period at 37uC, PGE2 production was determined by ELISA. Data in E and F represent the mean6S.D. of triplicate samples from a representative experiment of three with similar results. * = P,0.01. (G) HUVECs were treated with actinomycin D (AMD) (5 mg/ml) or vehicle and, after 45 min, were either subjected to heat shock (HS) at 43uC for 40 min or left untreated. At 1,5 hour during the recovery period at 37uC, total RNA was extracted and levels of COX-2 and b-actin mRNA were analyzed by real-time PCR. For real-time PCR (panels B and G), relative quantities of COX-2 RNAs were normalized to b-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate 6 S.D. Fold induction was calculated as described in Fig. 1. doi:10.1371/journal.pone.0031304.g002 after treatment. As shown in Fig. 2E, heat stress induced a time- dependent increase in PGE2 production. As early as 2 hours during the recovery period the increase in PGE2 production was comparable to that reached in HUVECs stimulated with IL-1b for 6 hours, despite the fact that higher levels of COX-2 were detected in IL-1b-treated cells. PGE2 levels continued to rise until 4– 6 hours after stress. Levels of the prostacyclin metabolite 6-keto PGF1a were also found to be increased in the same samples (Rossi A., personal communication). To determine whether heat-induced PGE2 production was dependent on de-novo synthesis of heat-induced COX-2 expression or was the consequence of increased prostanoid secretion, PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31304