The p110 and p110 Isoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in
Toll-Like Receptor 5 Signaling in Epithelial Cells
Figure 3
PI3K inhibition reduces flagellin-stimulated IL-8 promoter activation and mRNA production. (a) Caco-2 cells were electroporated with and IL-8 promoter/luciferase reporter plasmid and pEGFP as a transfection marker and grown as described in Methods. Cells were pretreated with inhibitors at the concentrations shown (or an equivalent volume of DMSO vehicle) followed 30 minutes later by flagellin 100 ng/ml. After 6 hours, cells were harvested for luciferase assay and fluorescence determination. The RLU/RFU value was calculated for each well, and these data normalized to the wells stimulated with DMSO + flagellin in each individual experiment. Data shown are compiled from at least 3 experiments for each inhibitor. (t-test). (b) IL-8 mRNA measurements by quantitative RT-PCR compiled from at least 3 experiments. Cytoplasmic RNA isolated from cells treated as shown was reverse transcribed. cDNA was amplified using actin and IL-8 primers with real-time fluorescent quantitation. Fold increases in mRNA were calculated as described in Methods. Results are expressed as the ratio compared to cells treated with flagellin plus DMSO vehicle. versus flagellin plus DMSO (t-test).
