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

Fig 1. vGPCR oncogene expression increases COX-2 mRNA and protein expression levels as well as COX-2 activity.

Fig 1

A) Fold-changes of COX-2 mRNA expression in transformed NIH3T3 (NIHT3T3-vGPCR) cells that stably express the vGPCR oncogene and control cells were assessed by RT-qPCR in triplicate and are presented as means ± SD. (*P <0.05). B) NIH3T3 cells were transfected with vGPCR expression vectors and were incubated ON in serum-free media. The vGPCR full agonist Gro-α (25nM), the COX-2 inhibitor NS398 (10uM), the ERK1/2 MAPK inhibitor PD98059 (20uM), or the p38 inhibitor SB220025 (10uM) were added to the cells as indicated. COX-2 activity was assessed measuring PGE2 production in the supernatants by an ELISA. Bars indicate mean PGE2 production of duplicate determinations ± SD. (*) Indicates significant differences between NIH3T3 control cells and the NIH3T3-vGPCR group of samples (P<0.05). (#) Indicates significant differences between sets of NIH3T3-vGPCR cells (P<0.05). C) Fold-changes of COX-2 mRNA expression in transformed SVEC (SVEC-vGPCR) cells that stably express the vGPCR oncogene and control cells were assessed by RT-qPCR in triplicate and are presented as means ± SD. (*P <0.05). D) COX-2 protein expression levels were determined by immunoblotting in SVEC cells that stably express the vGPCR oncogene. GAPDH was used as a loading control. COX-2 protein levels were measured in triplicate and are presented as means ± SD. (*P <0.05). E) SVEC cells were transfected with vGPCR expression vectors and were incubated ON in serum-free medium. The vGPCR full agonist Gro-α (25nM), the COX-2 inhibitor NS398 (10uM), the ERK1/2 MAPK inhibitor PD98059 (20uM), or the p38 inhibitor SB203580 (10uM) were added to the cells as indicated. COX-2 activity was assessed measuring PGE2 production in the cell supernatants by an ELISA. Bars indicate mean PGE2 production of duplicate determinations ± SD. (*) Indicates significant differences between samples from SVEC control cells and the SVEC-vGPCR group of samples (P<0.05). (#) Indicates significant differences between sets of SVEC-vGPCR cells (P<0.05). F) Total and phospho-ERK1/2 levels were determined by immunoblotting in SVEC cells transfected with vGPCR. The ERK1/2 MAPK inhibitor PD98059 (20uM), the p38 inhibitor SB203580 (10uM), the COX-2 inhibitor NS398 (10uM), or the vGPCR full agonist Gro-α (25nM) were added to the cells as indicated. Actin was used as loading control. pERK1/2 levels related to Total ERK1/2 levels were measured in triplicate and are presented as means ± SD. (*) Indicates significant differences between samples from SVEC control cells and the SVEC-vGPCR group of samples (P<0.05). (#) Indicates significant differences between sets of SVEC-vGPCR cells (P<0.05).