Figure 6
Figure 6. RasGRP1 restores G-CSF–dependent myeloid cell differentiation from Gfi1-null bone marrow progenitors. (A) Retrovirus-mediated expression of RasGRP1 or Gfi1 in 32D cells accelerates G-CSF–induced differentiation. The top panel shows representative nuclear morphology from DAPI staining of 32D cells during G-CSF incubation; for quantification, stages 1 and 2 were considered undifferentiated nuclei; stages 3 to 6 were considered differentiated nuclei. The bottom panel reflects the kinetics of G-CSF–induced differentiation of control 32D cells (parental, GFP, vector), and those transduced with RasGRP1 or Gfi1. Representative results from 3 experiments are shown. (B) Enhanced differentiation of Gfi1 or RasGRP1-transduced 32D cells after incubation with G-CSF for 3 days (5 experiments; the error bars reflect SDs). Cell populations are described in panel A. (C) Relative RasGRP1 expression in primary Gfi1−/− and Gfi1+/+ bone marrow MNCs 48 hours after retroviral transduction (RasGRP1 or vector only), no transduction (control) or mock transduction (Gfi1 mock) as assessed by real-time PCR. The results are representative of 5 independent transductions. (D) Effects of RasGRP1 transduction on G-CSF–induced activation of Erk1/2 in primary Gfi1-null bone marrow MNCs as assessed by immunoblotting with specific antibodies. After transduction, bone marrow MNCs were cultured for 72 hours in medium (Iscove DMEM with 10% FCS) supplemented with 10 ng/mL IL-3, 25 ng/mL SCF, and 5 ng/mL granulocyte-macrophage–CSF. Before G-CSF activation, the transduced cells were cytokine-starved by incubation for 2 hours in medium only. The bar graph reflects quantitative analysis of band intensities (phospho-Erk/total Erk) as measured by National Institutes of Health ImageJ software. Representative results are shown (of 3 performed). (E) Colony formation in semisolid methylcellulose medium supplemented with IL-3, IL-6, and SCF. Bone marrow MNCs transduced 72 hours earlier (vector only, RasGRP1 retrovirus, or mock-transduced) were cultured in methylcellulose for 14 days; the results reflect the number of colonies/dish (representative experiment of 4 performed). (F) Colony formation in semisolid methylcellulose medium supplemented with G-CSF. The cell populations (described in panel E) were transduced 72 hours before culture in methylcellulose for 12 days. The results reflect the mean number of colonies/dish (± SD) from 3 independent experiments. (G) G-CSF–induced myeloid cell differentiation in liquid cultures. The indicated cell populations were seeded 48 hours after transduction, mock transduction, or no transduction. After 4 days of incubation, cytospin preparations were stained with Giemsa. The left panel shows typical juvenile and mature neutrophils from G-CSF–stimulated primary bone marrow cells from Gfi1+/+ vector-transduced and Gfi1−/− cells transduced with RasGRP1. The right panel reflects the percentage of cells with juvenile and more mature granulocytic morphology as assessed microscopically by an independent observer. The results reflect the means ± SD from 3 independent experiments.

RasGRP1 restores G-CSF–dependent myeloid cell differentiation from Gfi1-null bone marrow progenitors. (A) Retrovirus-mediated expression of RasGRP1 or Gfi1 in 32D cells accelerates G-CSF–induced differentiation. The top panel shows representative nuclear morphology from DAPI staining of 32D cells during G-CSF incubation; for quantification, stages 1 and 2 were considered undifferentiated nuclei; stages 3 to 6 were considered differentiated nuclei. The bottom panel reflects the kinetics of G-CSF–induced differentiation of control 32D cells (parental, GFP, vector), and those transduced with RasGRP1 or Gfi1. Representative results from 3 experiments are shown. (B) Enhanced differentiation of Gfi1 or RasGRP1-transduced 32D cells after incubation with G-CSF for 3 days (5 experiments; the error bars reflect SDs). Cell populations are described in panel A. (C) Relative RasGRP1 expression in primary Gfi1−/− and Gfi1+/+ bone marrow MNCs 48 hours after retroviral transduction (RasGRP1 or vector only), no transduction (control) or mock transduction (Gfi1 mock) as assessed by real-time PCR. The results are representative of 5 independent transductions. (D) Effects of RasGRP1 transduction on G-CSF–induced activation of Erk1/2 in primary Gfi1-null bone marrow MNCs as assessed by immunoblotting with specific antibodies. After transduction, bone marrow MNCs were cultured for 72 hours in medium (Iscove DMEM with 10% FCS) supplemented with 10 ng/mL IL-3, 25 ng/mL SCF, and 5 ng/mL granulocyte-macrophage–CSF. Before G-CSF activation, the transduced cells were cytokine-starved by incubation for 2 hours in medium only. The bar graph reflects quantitative analysis of band intensities (phospho-Erk/total Erk) as measured by National Institutes of Health ImageJ software. Representative results are shown (of 3 performed). (E) Colony formation in semisolid methylcellulose medium supplemented with IL-3, IL-6, and SCF. Bone marrow MNCs transduced 72 hours earlier (vector only, RasGRP1 retrovirus, or mock-transduced) were cultured in methylcellulose for 14 days; the results reflect the number of colonies/dish (representative experiment of 4 performed). (F) Colony formation in semisolid methylcellulose medium supplemented with G-CSF. The cell populations (described in panel E) were transduced 72 hours before culture in methylcellulose for 12 days. The results reflect the mean number of colonies/dish (± SD) from 3 independent experiments. (G) G-CSF–induced myeloid cell differentiation in liquid cultures. The indicated cell populations were seeded 48 hours after transduction, mock transduction, or no transduction. After 4 days of incubation, cytospin preparations were stained with Giemsa. The left panel shows typical juvenile and mature neutrophils from G-CSF–stimulated primary bone marrow cells from Gfi1+/+ vector-transduced and Gfi1−/− cells transduced with RasGRP1. The right panel reflects the percentage of cells with juvenile and more mature granulocytic morphology as assessed microscopically by an independent observer. The results reflect the means ± SD from 3 independent experiments.

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