Tumor necrosis factor (TNF) and interleukin-1β (IL-1β) upregulate PU.1 protein in hematopoietic stem cells (HSCs) in vitro. (A) Examples of PU.1–enhanced yellow fluorescent protein (eYFP) expression kinetics in single HSCs upon TNF stimulation. Solid line: smoothed PU.1-eYFP expression dynamics from individual measurements (circles). Cell image tile length is 30 μm. (B) PU.1 induction by cytokine stimulation in HSCs. PU.1-eYFP fold changes were calculated for individual HSCs as 12 hours vs 0 hours values from continuous quantitative live imaging. Media was supplemented in all conditions with stem cell factor (SCF) (control) and additional cytokine as indicated. Analyzed HSCs SCF: 165, TPO: 255, IL-3: 112, IL-6: 89, interferon-γ (IFN-γ): 79, IFNα: 100, CSF3: 82, CSF2: 85, IL-1β: 94, TNF: 98, from n = 3-7 independent experiments/cytokine. ***P < 1 × 10−6, Bonferroni corrected analysis of variance (ANOVA). No other significant differences were detected. (C) PU.1-eYFP time courses of single cells cultured for 12 hours. Shown traces are sampled from 3 representative animals per condition. Control: 73, TNF: 101, IL-1β: 73. (D) PU.1-eYFP time-courses of single cells in presence of TNF + 50 µg/mL translation inhibitor cycloheximide. Cells: TNF: 86, TNF + cycloheximide: 27.