Fig. 3
From: Dynamic assessment of PI4P metabolism using split GFP based sensors
Stably expressed at low levels, the sGFP(PI4P) sensor combines a sensitive readout for OSW-1 dose response by FACS combined with high resolution spatial information. A Low magnification image of a population of HEK293 cells that stably express low levels of sGFP(PI4P) after exposure to various concentration of OSW-1 for 24 h. B The stability and intensity of the sGFP signal facilitates quantitative FACS analysis to obtain an OSW-1 dose response. To allow for a proper evaluation of shifts of the cell population based on GFP signal, standard plots that show absolute cell (event) numbers versus GFP signal were normalized (area under curve) to accommodate differences in total cell number before plot merger. The color coded OSW-1 concentrations are indicated C At an early time point, the average sGFP(PI4P) signal across a cell population does not provide a quantitative readout of PI4P increases (compare Fig. 2), but a confocal cross section view shows the onset of Golgi fragmentation. Classic markers (see mCherry-FAPP1 in Fig. 1D) show significant untargeted signal from excess, untargeted probe unless ideal expression levels are achieved. By comparison, the on-site sGFP signal creation for sGFP(PI4P) provides robust signal without significant untargeted GFP background.
