Live single-cell quantifications reveal heterogeneity in ERK signaling dynamics upon cytokine stimulation in human HSPCs. (A) Experimental scheme. The KTR-based fluorescent reporter is depicted to scale. (B) Schematic representation of the subcellular distribution of KTRs. When ERK is active, KTRs translocate from the nucleus to the cytoplasm, illustrated by representative video frames of a human HSC stimulated with 3F at indicated time. Shown are all 3 imaging channels and the overlay with the nuclear mask (scale bar, 10 μm). Gray bar at the top represents the timing of cytokine presence, and arrows indicate the timing of stimulation (media exchange) at 0 minutes and refreshment at 60 minutes. The same cell is shown in panel Cii and supplemental Video 2. (C) Single-cell time series and heat-stripe representation of ERK signaling response for 3 representative HSCs upon the stimulation with 3F. (D) Heat-stripe representation of ERK signaling dynamics in 90 single HSCs (3 independent experiments) in response to 3F. Each row corresponds to a single HSC, and cells are sorted based on cumulative ERK signal (no to high). Cells shown in panel C are highlighted in panels Di-iii). Two or 3 pooled UCB units were used per experiment. (E-F) Specificity of ERKKTR sensor. (E) Heat-stripe representation showing absence of ERK signaling in CD34⁺ cells treated with blank medium (no 3F, 33 cells) and 3F plus ERK inhibitors PD0325901 (10 µM, 40 cells) or U0126 (10 µM, 44 cells) from 2 independent experiments. (F) Quantification of anti-phospho-p44/42 immunostaining 1 hour after stimulation of cells in panels D and E. Unpaired Student t test with Welch’s correction. ***P < .0005; ns (not significant), P > .05. P2A , picornavirus 2A. SFFV, spleen focus-forming virus.