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. 2020 Oct 15;16(10):e1009006. doi: 10.1371/journal.ppat.1009006

Fig 2. vGPCR signaling regulates COX-2 promoter activity and induces mRNA stability via ERK1/2.

Fig 2

A) SVEC cells were transfected at increasing concentrations with a vGPCR expression vector and a luciferase reporter plasmid under the control of the COX-2 promoter. Luciferase activity expressed as fold induction relative to control cells that do not express vGPCR. Luciferase activity was measured in triplicate and is presented as means ± SD. (*P<0.05). B) Stably transfected SVEC-vGPCR cells and control cells were transfected with a luciferase reporter plasmid under the control of the COX-2 promoter. Luciferase activity was measured in triplicate and is presented as means ± SD. (*P<0.05) and expressed as fold induction relative to control cells. C) A reporter that expresses Luciferase under the control of a COX- 2 gene promoter region was co-transfected with a vGPCR expression vector and plasmids expressing constitutively active and dominant negative MAP kinase kinases (MEKEE and MEKAA respectively) or treated with the MEK/ERK1-2 inhibitor PD98059. Luciferase activity was tested and presented as fold induction relative to SVEC control cells. (*) Indicates significant differences relative to SVEC control untreated cells (P<0.05). (#) Indicates significant differences relative to SVEC-vGPCR untreated cells (P<0.05). D) mRNA stability assays were performed using a reporter plasmid containing the COX-2 3’UTR region cloned downstream of the luciferase ORF from SVEC or SVEC-vGPCR cells. Actinomycin D (5 μg/ml) was added (t = 0) to arrest transcription, and mRNA levels of Luciferase mRNA were analyzed by qRT-PCR following a time course (4 hours). Luciferase mRNA was measured in triplicate and is presented as means ± SD. (*P <0.05). E) mRNA stability assays in SVEC-vGPCR cells transfected with the same reporter plasmid as in D) in the presence or absence of the MEK/ERK1-2 inhibitor PD98059 (20 uM). Actinomycin D (5 μg/ml) was added (t = 0) to arrest transcription, and mRNA levels of Luciferase mRNA were analyzed by qRT-PCR following a time course (4 hours). Luciferase mRNA was measured in triplicate and is presented as means ± SD. (*P<0.05).