FIG 5.

Identification of BoHV-1 intergenic regions that are regulated by stress-induced transcription factors. (A) Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene that contains the BoHV-1 sequences, a plasmid that expresses the GR plasmid (1 μg of DNA), and a plasmid that expresses Renilla luciferase (0.05 μg of DNA). Following transfection, Neuro-2A cells were cultured in 2% stripped fetal calf serum after transfection. Twenty-four hours after transfection the designated Neuro-2A cultures were treated with water-soluble DEX (10 μM; Sigma). (B) Neuro-2A cells were cotransfected with the designated plasmid constructs (0.25 μg of DNA) containing the firefly luciferase gene and viral sequences, a plasmid encoding Renilla luciferase (0.05 μg of DNA), and a KLF transcription factor (KLF4, KLF15, or promyelocytic leukemia zinc finger [PLZF]) or a plasmid expressing Slug-1 (0.5 μg of DNA). The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation values for other samples are presented. At 48 h after transfection, cells were harvested, and protein extracts were subjected to a dual-luciferase assay as described in the Materials and Methods section. The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation values for other samples are presented. The results are the average of three independent experiments, and error bars denote the standard errors.