Fig. 1. Estimation of [cAMP]_i changes using FRET in NCI-H292 cells. NCI-H292 cells were co-infected with HIV-derived lentiviruses encoding the fluorescently labeled subunits of PKA driven by the CMV promoter. (A) FRET was demonstrated by exciting cells at 436 nm and visualizing CFP emission at 480 nm (left panel, blue) and YFP emission at 535 nm from a pair of cells (middle panel, green). Right panel shows merged image. Bar, 10 µm. (B) Repeated stimulation of the two cells pictured in A (left cell, thin lines; right cell, thick lines) with 20 µM forskolin increased CFP (blue trace) and decreased YFP emission (green), consistent with a reduction in FRET due to separation of the PKA subunits. (C) Ratio data (CFP:YFP fluorescence; left cell: thin line and right cell, thick line) reveal reversible increases in estimated [cAMP]_i upon exposure to 20 µM forskolin.