Extended Data Fig. 3: Multi-dimensional analyses comparing EKAREV, EKAREN4 and EKAREN5.
a, FRET-range versus sensor expression level, as in Extended Data Fig. 2e (EKAREN4, n=80 cells; EKAREN5, n=75 cells). a’, baseline and plateau ratios corresponding to cells plotted in a. b, Means (± s.d.) of ΔR from a, calculated in three expression level categories (as in Extended Data Fig. 2g). c, Dephosphorylation kinetics of EKAREN5 were directly compared with EKAREV(Tq) (as Extended Data Fig. 2d). Retrospective unmixing was based on clustering plateau amplitudes (PMA) (see Fig. 2a). Experiment performed once. d, As in c, comparing phosphorylation and dephosphorylation kinetics of EKAREN5 with ~33-fold expression level difference. Co-seeded high and low expressors were simultaneously monitored. Experiment performed three times. e-h, Various automated analyses on autonomous ERK fluctuations of HeLa cells (dataset of Fig. 2f,g), registered simultaneously by ERK-KTR-mCherry and either of EKAREV/EKAREN FRET sensors. EKAREV, n=15 single-cell traces; EKAREN4, n=10 single-cell traces; EKAREN5, n=17 single-cell traces. e, Automated peak counting per individual cell. f, Temporal matching of rising phases in KTR versus FRET signals. g, Temporal matching of falling phases in KTR versus FRET signals. h, Counted ‘inflection’ points per trace, that is points where ERK changes accelerate or decelerate(see Methods). i, Correlation between EKAREN5-FRET and ppERK staining (mean nuclear signal). After various ERK manipulations, HeLa-EKAREN5 cells were FRET-imaged and fixed instantly after acquisition, yielding various ERK activity states between complete inhibition (MEKi+ERKi) and pathway saturation (>7 min EGF). Grey line, regression analysis (y=ax+b). Traces are mean ratios ± s.d. For panels a-i the n numbers represent cells and are indicated in the graph for each group. Box-and-whisker plots: boxes represent quartile 2 and 3, horizontal line represents median, whiskers represent minimum and maximum within 1,5x interquartile range. Dots, outliers. Scale bar, 50μm. Two-sided student’s T-tests: *, p<0.05; **, p<0.005; n.s., non-significant.
