Fig. S3

Fig. S3 JNK KTR Enables Single-Cell Measurements of Kinase Activity Dynamics, Related to Figure 2>

(A) 3T3 cells expressing JNK KTR (wild-type or with phospho sites mutated to alanine, AA, or glutamic, EE) were stimulated with anisomycin (50 ng/ml) and imaged at indicated time points. Where indicated (+ JNK inh. or + p38 inh.), cells were preincubated for 45 min with 10 ?M JNK inhibitor VIII or 10 ?M SB203580. Representative cells are shown for each construct or condition over time.

(B) Heat maps of data presented in Figure 1G are shown. Data represent more than 100 cells for each condition, obtained from 3 independent experiments.

(C) 3T3 cells were stimulated with IL-1? (1 ng/ml) and harvested at indicated time points for quantitative Western blot (WB) analysis. Representative of 3 independent experiments.

(D) JNK KTR cells were stimulated with IL-1? (1 ng/ml), imaged and quantified as described in Methods. Three independent experiments were performed resulting in 980 single cells measured. KTR data represent the mean ± SD from the 3 experiment means (averaged to mimic in silico WBs). WB data are calculated as the fraction of phosphorylated over total and represents the mean ± SD from 3 independent experiments. All data sets were normalized between 0 and 1 for comparison.

(E) 3T3 JNK KTR cells were stimulated with anisomycin (50 ng/ml) for 0 or 20 min and fixed with 4% PFA for immunofluorescence (IF) analysis. Phospho-JNK (left) and phospho-c-Jun(S63) (right) antibodies were detected using a Cy5-linked secondary antibody. 10 ng/ml of DAPI was used to stain the nucleus. Representative cells are shown for each time point.

(F) 3T3 JNK KTR cells were stimulated with IL-1? (1 ng/ml) for indicated times and fixed with 4% PFA for quantitative IF analysis. 10 images were taken for each time point and quantified as described in Methods. For each cell C/N KTR ratio (red) and phospho-Jun intensity (black) were determined. All data sets were normalized between 0 and 1 for comparison. Data represent the mean ± SD from more than 500 cells for each time point obtained from 2 independent experiments. IF data are overlaid on the dynamic JNK KTR data set (blue). Note that in this case, JNK KTR dynamic data represent the mean ± SD from all individual cells (n = 980), obtained in 3 independent experiments.

(G) IF data obtained in Panel b represented as contour scatter plot. Single cell JNK KTR ratio and phospho-Jun intensity from all time points are shown. Contour color represent areas of increasing data point density. Raw scatter plots fitted to a linear regression are shown together with R and P values.

(H) JNK KTR cells were imaged at a single time point and quantified as described in Methods. Correlations of expression level with basal C/N ratio are shown. Data represent 248 cells obtained from 3 independent experiments.

(I) JNK KTR cells were stimulated with IL-1? (1 ng/ml), imaged and quantified as described in Methods. Peaks were identified using custom software. Correlations between fold ratio induction and expression level are shown. Data represent 92 cells obtained from 2 independent experiments.

(J) Indicated cell lines expressing JNK KTR were stimulated with anisomycin (50 ng/ml) and imaged at indicated time points. Representative cells are shown for each cell line over time.