Figure 3
Figure 3. Oncogenic Nras signaling engages myeloid progenitors into cell cycle and leads to their expansion in a dose-dependent manner. Different tissues were isolated and analyzed 2 days after pI-pC injections. (A) Quantitative analysis of myeloid progenitor (MP) compartment in bone marrow and spleen of control, Nras G12D/+, and Nras G12D/G12D mice. CMP indicates common myeloid progenitor; GMP, granulocyte-monocyte progenitor; and MEP, megakaryocyte-erythroid progenitor. Results are presented as averages + SDs. Student t test was performed: *P < .05, and **P < .01. (B) Cell cycle analysis of MPs in bone marrow of control, Nras G12D/+, and Nras G12D/G12D mice. Cell-cycle phases are defined as G0 (Ki67−, DAPIlo), G1 (Ki67+, DAPIlo), and S/G2/M (Ki67+, DAPIhi). The percentages of MPs in individual cell-cycle phases are indicated on the density plots. Average values + SDs are shown in the right graph. Student t test was performed: *P < .05. (C,D) Phospho-flow analysis of p-ERK1/2 in Lin−/low c-Kit+ bone marrow cells of control, Nras G12D/+, and Nras G12D/G12D mice 2 days after the second pI-pC injection. (C) Total bone marrow cells were freshly isolated and stimulated with or without 2ng/mL of GM-CSF at 37°C for 10 minutes. Basal condition is defined as without GM-CSF stimulation. U0126 was mixed with cells for 30 minutes before fixation or GM-CSF stimulation. Levels of p-ERK1/2 were measured using phospho-specific flow cytometry. Nonneutrophil Lin−/low c-Kit+ cells were gated for data analysis. Results obtained from one representative experiment are shown (left panel). Quantification of 6 independent experiments is shown as average values + SDs (middle panel). Solid lines indicate the median intensity of p-ERK1/2 in control cells without GM-CSF stimulation (right panels). (D) Total bone marrow cells were serum- and cytokine-starved for 1 hour and stimulated with various concentrations of GM-CSF (0, 0.16 and 2 ng/mL) at 37°C for 10 minutes. Gating strategy and plots of p-ERK1/2 are representative of 4 independent experiments. To quantify the activation of ERK1/2, median intensities of p-ERK1/2 at different GM-CSF concentrations in different animals are compared with control cells at 0 ng/mL, which is arbitrarily set at 1. Average values ± SDs are shown in the right graph. Student t test was performed: *P < .05.

Oncogenic Nras signaling engages myeloid progenitors into cell cycle and leads to their expansion in a dose-dependent manner. Different tissues were isolated and analyzed 2 days after pI-pC injections. (A) Quantitative analysis of myeloid progenitor (MP) compartment in bone marrow and spleen of control, Nras G12D/+, and Nras G12D/G12D mice. CMP indicates common myeloid progenitor; GMP, granulocyte-monocyte progenitor; and MEP, megakaryocyte-erythroid progenitor. Results are presented as averages + SDs. Student t test was performed: *P < .05, and **P < .01. (B) Cell cycle analysis of MPs in bone marrow of control, Nras G12D/+, and Nras G12D/G12D mice. Cell-cycle phases are defined as G0 (Ki67−, DAPIlo), G1 (Ki67+, DAPIlo), and S/G2/M (Ki67+, DAPIhi). The percentages of MPs in individual cell-cycle phases are indicated on the density plots. Average values + SDs are shown in the right graph. Student t test was performed: *P < .05. (C,D) Phospho-flow analysis of p-ERK1/2 in Lin−/low c-Kit+ bone marrow cells of control, Nras G12D/+, and Nras G12D/G12D mice 2 days after the second pI-pC injection. (C) Total bone marrow cells were freshly isolated and stimulated with or without 2ng/mL of GM-CSF at 37°C for 10 minutes. Basal condition is defined as without GM-CSF stimulation. U0126 was mixed with cells for 30 minutes before fixation or GM-CSF stimulation. Levels of p-ERK1/2 were measured using phospho-specific flow cytometry. Nonneutrophil Lin−/low c-Kit+ cells were gated for data analysis. Results obtained from one representative experiment are shown (left panel). Quantification of 6 independent experiments is shown as average values + SDs (middle panel). Solid lines indicate the median intensity of p-ERK1/2 in control cells without GM-CSF stimulation (right panels). (D) Total bone marrow cells were serum- and cytokine-starved for 1 hour and stimulated with various concentrations of GM-CSF (0, 0.16 and 2 ng/mL) at 37°C for 10 minutes. Gating strategy and plots of p-ERK1/2 are representative of 4 independent experiments. To quantify the activation of ERK1/2, median intensities of p-ERK1/2 at different GM-CSF concentrations in different animals are compared with control cells at 0 ng/mL, which is arbitrarily set at 1. Average values ± SDs are shown in the right graph. Student t test was performed: *P < .05.

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