Supplementary Figure 6: Characterization of single-fluorophore kinase sensors in cultured rat cortical neurons and HeLa cells.
From: Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities
(a) Average time-course of ExRai-AKAR (Ex488) and dsRed responses in the cell soma following Fsk stimulation. Insets: Representative ExRai-AKAR fluorescence images before (-Fsk) and after (+Fsk) stimulation and maximal responses (F/F0) (****p<0.0001, two-tailed paired Student’s t-test; n = 19 neurons pooled from 3 experiments]). Data are shown as dot plots; bars represent mean ± SEM. (b) Average time-course of RAB-EKARev and GFP responses in the cell soma following DHPG stimulation. Insets: Representative RAB-EKARev fluorescence images before (-DHPG) and after (+DHPG) stimulation and maximal responses (F/F0) (P = 0.0117, paired Student’s t-test; n = 6 neurons pooled from 3 experiments). Data are shown as dot plots; bars represent mean ± s.e.m. (c) Average time-course of ExRai-AKAR (Ex488), RAB-EKARev, and BCaMP responses in the cell soma following Fsk stimulation. Inset: maximal responses (F/F0) of ExRai-AKAR, RAB-EKARev, and BCaMP (p = 0.0005, ****p<0.0001, Kruskal-Wallis test; n = 68 neurons pooled from 6 experiments). (d) Average time-course of ExRai-AKAR (488 nm excitation), RAB-EKARev, and BCaMP responses in the cell soma following DHPG stimulation. Inset: Maximal responses (F/F0) of ExRai-AKAR, RAB-EKARev, and BCaMP (****p<0.0001, Kruskal-Wallis test; n = 104 neurons pooled from 10 experiments). Box-and-whisker plots depict the median, interquartile range, min, max, and mean (+). All data points are shown. Maximum responses are calculated with respect to the initial intensity at time 0 (F/F0). (e) Domain structures of Lyn-sapphireAKAR and sapphireAKAR-NLS. (f, g) Average time-courses of (f) Lyn-sapphireAKAR (n = 5 cells) and (g) sapphireAKAR-NLS (n = 6 cells) responses in HeLa cells treated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX). (h) Domain structure of Lyn-RAB-EKARev and RAB-EKARev-NLS. (i, j) Average time-courses of (i) Lyn-RAB-EKARev (n = 9 cells) and RAB-EKARev-NLS (n = 18 cells) in HeLa cells treated with 100 ng/mL EGF. Data in f, g, i, and j are pooled from two independent experiments; representative images show probe localization. Curves are normalized with respect to the average baseline intensity (a-d) or to time 0 (F/F0) (f, g, i, and j). Solid lines represent the mean; shaded areas, SEM. Scale bars, 10 μm. See Supplementary Table 1 for bar graph source data
