Fig. 5

Live cell imaging in Flamindo2-expressing MN9D cells demonstrates that PDE2A inhibition leads to increased levels of cAMP. A Schematic representation of the domain structure of Flamindo2. Citrine is a mutant of yellow fluorescent protein (YFP). Flamindo2 is created by insertions of DNA fragments that encode the cAMP binding domain of mEPAC2. B Schematic working mechanism of Flamindo2. Binding of cAMP to the cAMP binding domain reduces fluorescence of the Flamindo2 protein. Therefore, decreasing fluorescence indicates elevations in cAMP levels within a cell. C Experimental timeline of the live cell imaging experiments in Flamindo2 expressing MN9D cells. Prior to application of vehicle, forskolin, or phosphodiesterase (PDE) inhibitors, a baseline measurement in fluorescence intensity of 100 s was performed. Images were collected every 20 s. D–G Representative images showing fluorescence intensity just before application of the compounds at 0 s, at 20 s of incubation with the compound, and after 100 s. Representative image are shown for treatment with a vehicle (D), 10 µM forskolin (E), 10 µM BAY 60-7550 (F), and 100 µM PF05180999 (G). H–J 95% confidence intervals time-courses representing the changes in fluorescence intensity induced by forskolin (H), BAY 60-7550 (I), or PF05180999 (J). Upper graphs show change in fluorescence intensity relative to the baseline period (∆F/F), while lower graphs are photobleaching effect-corrected representations of the percentual change in fluorescence intensity of each compound as compared to their vehicle