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Article Figures & Data

Figures

  • Figure 1.

    Transduction of murine bone marrow cells and ectopic Shp-2 expression. (A) Low-density bone marrow mononuclear cells from C57/Bl6 mice were transduced with ecotropic retroviral supernatants (pMIEG3, pMIEG3-WT, pMIEG3-E76K, pMIEG3-D61V, and pMIEG3-D61Y) and tranduced cells were enriched by sorting for EGFP expression. Representative FACS showing the percentage of EGFP+ cells for collection of transduced cells is demonstrated. (B, left) Western blot analysis demonstrated that the exogenously introduced Shp-2 proteins are equally expressed in each group of transduced cells. Shp-2 band intensities normalized to GAPDH are shown graphically (right). Representative Western blot of 2 independent experiments with similar results.

  • Figure 2.

    PTPN11 mutants E76K, D61V, and D61Y induce hematopoietic progenitor hypersensitivity to GM-CSF. Transduced EGFP+ cells were plated in CFU-GM progenitor assays in duplicate in 3 independent experiments. The data are represented as a percentage of maximal colony formation, which is calculated by dividing the number of colonies at each dose by the number of colonies at the maximal GM-CSF concentration (10 ng/mL). Error bars indicate standard error of the mean. Statistical analysis was conducted using random-effects ANOVA to deal with repeated measurements from duplicate plates. (A) E76K vs MIEG3 and WT; *P = .0014 for E76K vs MIEG3 and *P = .0015 for E76K vs WT; **P < .0001 for E76K vs MIEG3 or WT. (B) D61V vs MIEG3 and WT; *P < .0001 for D61V vs MIEG3 or WT. (C) D61Y vs MIEG3 and WT; *P = .0179 for D61Y vs MIEG3 and *P = .02 for D61Y vs WT; **P = .0058 for D61Y vs MIEG3 or WT; ***P < .0001 for D61Y vs MIEG3 or WT.

  • Figure 3.

    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.

  • Figure 4.

    Macrophage progenitors derived from MIEG3-, WT-, E76K-, D61V-, and D61Y-transduced cells express similar levels of the GM-CSF and M-CSF receptors. Given the variable response observed in macrophage proliferation in response to GM-CSF and M-CSF, receptor levels for each of these cytokines was examined. (A) Protein extracts prepared from macrophage progenitor cultures were evaluated for the unique α chain of the GM-CSF receptor. (B) Macrophage progenitors were collected, incubated with anti–M-CSFR, and secondarily stained with PE-conjugated F(ab′)2 fragment goat anti–rat IgG followed by FACS analysis to determine the percentage of M-CSFR+ cells.

  • Figure 5.

    PTPN11 mutants E76K, D61V, and D61Y confer constitutively elevated and prolonged GM-CSF–stimulated phospho-Erk levels in macrophage progenitors. (A) Representative Western blot showing 0- and 5-minute time points of GM-CSF stimulation of macrophage progenitor cultures; repeated in 3 independent experiments for MIEG3, WT, E76K, and D61V; repeated in 2 independent experiments for E61Y. (B) Band intensities were quantitated using densitometry, phospho-Erk band intensities were normalized to Erk band intensities, and data were compiled from all experiments and demonstrated graphically. Data shown as ± SEM; *P < .05 for E76K, D61V, and D61Y compared with MIEG3 at baseline. No statistical difference between MIEG3 and WT at baseline. (C) Western blot analysis demonstrating phospho-Erk levels following GM-CSF stimulation for 10 and 30 minutes demonstrating prolonged activation of Erk in macrophage progenitors transduced with each of the Shp-2 mutants. (D) A second independent experiment showing prolonged activation of Erk in macrophage progenitors transduced with each of the Shp-2 mutants for as long as 60 minutes following GM-CSF stimulation.