Fig. 2
From: Dynamic assessment of PI4P metabolism using split GFP based sensors
The sGFP(PI4P) sensor lacks the rapid response capability of its ddRFP(PI4P) counterpart, but shows superior sensitivity. A Time course for the response of HEK293 cells transfected transiently with ddRFP(PI4P) or sGFP(PI4P) following stimulation with 500nM OSW-1. The relative changes for three individual time courses (3–4 cells per observation window and time course) were merged. Within the two hours of observation, overexpressed ddRFP(PI4P) shows a clear response while no clear signal change emerges from sGFP(PI4P) at this time scale, as expected due to folding and fluorophore maturation delay. B Design of HA-tagged sensor constructs. Under the selected cotransfection conditions, both sensors are expressed at comparable levels on a population scale. Traces of HA-signal with an apparent molecular weight close to 100 kDa (*) are found also in HA-ddRFP(PI4P) only transfections and represent traces of fusion proteins with T2A read-through. C) At matched expression levels, sGFP(PI4P) shows a significantly stronger signal at normalized fluorescence detection conditions. D + E A quantitative assessment of a larger double transfected cell population shows that, even under conditions of overexpression, the sGFP(PI4P) sensor shows an approximately 60x higher signal. F At the high levels of transient expression used for the assessment of relative signal intensity in C-E, both sensors impact proliferation and cell adhesion negatively compared to a control transfection with mCherry. The relative count of viable attached and detached cells was measured by alamarBlue at day four post transfection. The combined signal from attached and detached cells was normalized for the mCherry control.
