PTPN11 mutants E76K, D61V, and D61Y induce macrophage progenitor hyperproliferation in response to GM-CSF but not to M-CSF. (A) Sorted EGFP⁺ cells subjected to macrophage progenitor differentiation conditions were stained with anti–Mac-1 or with anti-F4/80 and analyzed using FACS. Representative FACS analysis showing the percentage of Mac-1⁺ or F4/80⁺ cells is depicted; 2 independent experiments conducted with consistent results. (B) Macrophage progenitor cultures were serum- and growth factor–deprived for 24 hours and then stimulated with GM-CSF or M-CSF. Data shown as ± SEM. For pMIEG3, WT, E76K, and D61V, 3 independent experiments with each condition in triplicate were conducted; for D61Y, 2 independent experiments with each condition in triplicate were conducted. Statistical analysis was conducted using random-effects ANOVA to deal with repeated measurements from triplicate conditions; ^*P < .0001 for E76K vs MIEG3 or WT; ^**P = .0002 for D61V vs MIEG3 and ^**P = .0014 for D61V vs WT; ^***P = .019 for D61Y vs MIEG3 and ^***P = .074 for D61Y vs WT; #P < .0001 comparing E76K, D61V, or D61Y to MIEG3 or WT following GM-CSF treatment. No statistical difference was observed between cultures following stimulation with M-CSF.