AhR triggers Gα
/calcium/COX-2 signaling. (A) BBP induced a reduction in the level of PIP2 and activation of IP3R via Gαq/11. After Huh7 cells treated with BBP (1 μM), protein levels were analyzed by immunoblotting at the indicated time points. (B) Real-time imaging of calcium was performed by Cell-R microscopy. The experiment was performed in BSS medium. (C) Elevated intracellular calcium was induced by BBP treatment. The experiment was performed with a calcium free medium. The arrows indicate the time points of BBP (1 μM) addition (1 minute after the experiment started). The fluorescence intensity (Y-axis) indicates the relative calcium levels. (D) Huh7 cells in calcium-free medium were pretreated with various concentrations of 2-APB for 30 minutes before stimulation with BBP (1 μM). Internal calcium release was inhibited by 2-APB in a dose-dependent manner. The relative intensity of fluo-4 indicates the calcium levels: peak/baseline ratio of fluorescence intensity. Calcium-free medium was used for each experimental interval. Each value in the graph is the mean ± SD of six replicate using at least ten cells. (E) COX-2 expression after BBP treatment was measured by immunoblotting. Huh7 cells were treated with BBP (1 μM) for the indicated times before harvesting the cells. β-actin was used as an internal control. (F) Huh7 cells were pretreated with 2-APB (20 μM) for 2 hours and followed by treated with BBP (1 μM). Control groups were treated with DMSO. COX-2 levels were suppressed by 2-APB pretreatment.