Diminished COX-2/PGE2-Mediated Antiviral Response Due to Impaired NOX/MAPK Signaling in G6PD-Knockdown Lung Epithelial Cells
Fig 3
G6PD knockdown dysregulates the activation of c-JUN and NF-κB signaling.
(A) Scramble control and G6PD-kd A549 cells were pretreated with Tanshinone IIA (TSIIA), AP-1 inhibitor for 2 h and then treated with 15 ng/ml TNF-α for 3 h. The expression level of COX-2 was assessed by western blotting assay. β-Actin expression was shown as the loading control. Numbers represent relative fold differences of protein levels on the basis of densitometer quantitation. Data are means ±SD of three separate experiments, *,#p<0.05 indicate significant difference between cells with or without inhibitor pretreatment upon TNF-α stimulation. (B) The expression level of COX-2 was determined by western blot under 15 ng/ml TNF-α stimulation or combined with pre-treatment of NF-κB inhibitor, Helenalin, for 3 h in scramble control and G6PD-kd A549 cells. β-Actin expression was shown as the loading control. Numbers represent relative fold differences of protein levels on the basis of densitometer quantitation. Data are means ±SD of three separate experiments, *,#p<0.05 indicate significant difference between cells with or without inhibitor pretreatment upon TNF-α stimulation. (C) The phosphorylation level of c-JUN was determined by western blot in scramble control and G6PD-kd A549 cells stimulated with 1.5 or 15 ng/ml TNF-α in different time courses. β-Actin expression was shown as the loading control. Numbers represent relative fold differences of protein levels on the basis of densitometer quantitation. Data are means ±SD of three separate experiments, *p<0.05 indicates significant difference between scramble control and G6PD-kd cells upon TNF-α treatment at the corresponding time points. (D) The phosphorylation levels of p65 and IκBα were investigated in scramble control and G6PD-kd A549 cells upon TNF-α treatment (15 ng/ml) in different time courses. β-Actin expression was shown as the loading control. Numbers represent relative fold differences of protein levels on the basis of densitometer quantitation. Data are means ±SD of three separate experiments, *p<0.05 and **p<0.01 represent levels of significant difference between scramble control and G6PD-kd cells upon TNF-α treatment at the corresponding time points.