1. Initial testing and characterization of cAMPr in ES cells.
Initial testing and characterization of cAMPr in ES cells. (A) Overview of the screening process in embryonic stem (ES) cells. Forskolin was added to ES cells expressing the cAMP sensor variants, and those colonies that exhibited an increase in fluorescence were selected and sequenced. The sequences of the linkers of the forskolin-responsive clones are shown. (B) Left: Response of ES cell colonies expressing cAMPr to 40 μM forskolin (red; n = 15 colonies) or dimethyl sulfoxide (DMSO) vehicle (blue; n = 16 colonies) from two independent experiments. The gray box indicates the time of drug addition. Light red and blue shading represents the SEM. Data are normalized to the fluorescence at time zero. Right: Images show representative single ES colonies before (Pre; left) and after (Post; right) the application of 40 μM forskolin (top) or DMSO (bottom). P was calculated using a standard unpaired two-sided t test. (C) Response of cAMPr in digitonin-permeabilized ES cells to 1 mM cAMP or 1 mM cGMP. Left: Graph shows the average of at least 13 colonies from two independent experiments, and the data were plotted as described in (B). Right: Bar graph showing the maximum fold changes in fluorescence plotted for 1 mM cAMP (red) and 1 mM cGMP (Blue). (D) Response of ES cell colonies expressing cAMPr (left) or cAMP Difference Detector in situ (cADDis) (right) to the indicated concentrations of forskolin. Cells expressing cADDis that were exposed to DMSO are shown in black. Data are from 14 to 20 colonies from two independent experiments for all treatments. Data are plotted as described in (B). (E) Maximum average (max avg) fold change in fluorescence in response to the indicated concentrations of forskolin in ES cells expressing cAMPr (left) or cADDis (right) normalized to the maximum response to 40 μM forskolin using data from (D). Two-sample unpaired t tests for cAMPr showed P < 0.05 for each value plotted compared to the next highest concentration (40 μM versus 10 μM, P < 0.05; 10 μM versus 1 μM, P < 0.001; 1 μM versus 100 nM, P < 0.001; 100 nM versus 10 nM, P < 10−5). For cADDis, two-sample unpaired t tests only showed statistical significant differences for 1 μM versus 100 nM when compared to the next highest value (40 μM versus 10 μM, P = 0.7; 10 μM versus 1 μM, P = 0.5; 1 μM versus 100 nM, P < 10−8, 100 nM versus 10 nM, P = 0.10; 10 nM versus DMSO, P = 0.44).
Christopher R. Hackley et al., Sci. Signal. 2018;11:eaah3738
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