Transcriptional Regulation of CDC37 Promoter

The CDC37 promoter-luciferase reporter constructs (500+UTR and 200+UTR) [60 (link)], the overexpression constructs of heat shock factor 1 (HSF1) and dominant-negative (DN)-HSF1 [74 (link)], plasmid DNA co-transfection, and luciferase assay were described previously [60 (link),74 (link),78 (link)]. Briefly, cells were cultured in 96-well plates and a plasmid (25 ng reporter, 100 ng effector) was transfected with 0.4 µL FuGENE HD (Roche, Basel, Switzerland) per well at a cell confluence level of 50%–70%. The medium was changed at 16–20 h after transfection. At 40–48 h after transfection, 70 µL of the medium was aspirated, then 30 µL of Bright-Glo reagent (Promega, Madison, WI, USA) was added and mixed by pipetting. Cells were incubated for 5 min at 37 °C. The lysate (40 µL) was transferred to a 96-well white plate for measurement of luminescence.