Figure 6.
Sensing dynamics with repeated stimulation and live cell NFAT-EFP imaging. (a) HeLa cells transduced with Ad GnRHR and Ad NFAT-EFP were stained with Hoechst dye transferred to live cell imaging medium and imaged at 37°C both before and during stimulation with 0, 10−11, 10−9, or 10−7 M GnRH for 15 minutes (first gray bar). The plate was then removed from the stage, cells were washed (with PBS, three times during 5 minutes), and the plate was returned to the stage. The cells were imaged for a further 2 hours before repeat stimulation (second gray bar) for 60 minutes using the same concentrations of GnRH. Image analysis and data processing were as described under Fig. 4. The data shown are pooled from the tracked cells in three repeated experiments (mean ± standard error of the mean, n = 67 to n = 489). (b) MI values calculated using the AUC for the first 15 minutes of stimulation with GnRH in pulse 1 or pulse 2, the additional information gained by sensing both, and the MI between responses in pulse 1 and pulse 2. Note that these data are consistent with the stable effector scenario of Fig. 5, with little additional information gained by sensing both pulses because responses in one pulse are highly predictive of those in the other.