Figure 4.

PGE₂ levels and cPLA₂ activity are low in patients' fibroblasts, and could be rescued. (A) Levels of PGE₂ in cell culture media after 24 h of stimulation with LPS or cholera toxin (CT). Levels of PGE₂ were normalized against protein concentrations in the supernatants. All cells were primary human fibroblasts except RAW 264.7 cells, which are murine macrophage like cells and used as a positive control. (B) Activity of cPLA₂ in the membrane fractions of fibroblasts and RAW 264.7 macrophages. Cells were stimulated with or without LPS for 24 h before harvesting and purification of the membrane fractions. The cPLA₂ activity was normalized to amount of proteins added to the assay. (C) PGE₂ levels in the cell culture media after transfection with CMV promoter-based pIRES2-DsRed2 plasmid containing the native PLAA gene and a fluorescent marker of transfection. Cells were treated as follows: ctl = no transfection; V = transfection with empty vector; PLAA = transfection with plasmid vector containing the wild-type or native PLAA. (D) cPLA₂ activity from membrane fractions of fibroblasts after transfection with a plasmid containing the nPLAA in a CMV promoter-based vector system and a fluorescent marker of transfection. (E–G) Fold-changes in transcripts for IL6, IL8, and MIF based on RT-qPCR. Arithmetic means ± SD from three independent experiments performed in triplicate are plotted and the data analysed using one-way ANOVA with Tukey post hoc correction.