Increased expression of CD123 is associated with enhanced proliferation and IL-3R signaling in response to low concentrations of IL-3. TF-1.8 CD123-low (EV) or CD123-high-WT cells were starved of cytokine for 16 hours. A total of 105 cells were stimulated in a titration of human IL-3 (Ai) or GM-CSF (Aii) for 48 hours. Proliferation was measured by the addition of 0.5 µCi of 3H-thymidine/well for the final 5 hours of stimulation. Data are represented as the mean counts per minute ± standard deviation and is representative of 3 experiments. (B) Cells were washed and plated out at 5 × 105 cells/mL in medium containing IL-3 as shown on graph. After 48 hours, cells were costained with annexin V and propidium iodide and analyzed by flow cytometry. The average of 2 experiments each with triplicate samples with the standard error is indicated. (C) Cells were combined in a 99:1 ratio (99% TF-1.8 parental, 1% TF-1.8 CD123-high-WT) and cultures maintained in 0.1 ng/mL of IL-3 (Ci) or GM-CSF (Cii). The relative proportion of each population was monitored by flow cytometry. (D) Cells were starved of cytokine in media containing 0.5% FBS for 16 hours and stimulated with a titration of human IL-3 (Di) or GM-CSF (Dii) for 10 minutes at 37°C. The cells were lysed and human βc was immunoprecipitated, run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the phosphorylation of the tyrosine residues on the βc analyzed by western blotting, probing with anti-phospho-tyrosine mAb (clone 4G10). (E) Cells were starved and stimulated with IL-3 (Ei) or GM-CSF (Eii) as before. Whole cell lysates were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted. Blots were probed with antibodies to phosphorylated STAT-5 (Tyr694), Erk1/2 (Thr202/Tyr204), and Akt (Ser473) then reprobed with α-tubulin as a loading control. Statistical analysis: #nonsignificant difference, *P < .05, **P < .01, ***P < .001. Solid blue lines/bars, CD123-low (EV or parental cells); hashed or solid red lines/bars, CD123-high-WT.
