Figure 1
Figure 1. Erk activation is selectively defective in G-CSF–stimulated bone marrow cells from Gfi1-null mice. (A) G-CSFR mRNA levels in fresh (left) bone marrow MNCs from untreated Gfi1+/+, Gfi1+/−, and Gfi1−/− mice and after 24-hour incubation in vitro with or without G-CSF (25 ng/mL; right) measured by quantitative real-time PCR. (Left) Gfi1+/+ (n = 3); Gfi1+/− (n = 8); Gfi1−/− (n = 7); the results reflect the means ± SD. (Right) Means (duplicate measurements) of G-CSFR mRNA levels in bone marrow MNCs from individual mice. (B) Proliferation of bone marrow MNCs from Gfi1+/+, Gfi1+/−, and Gfi1−/− mice after 3-day incubation in medium only, with IL-3 (10 ng/mL), or with G-CSF (25 ng/mL). The results reflect the mean 3H thymidine uptake (cpm) of triplicate cultures ± SD and are representative of 3 independent experiments. (C) Immunoblot analysis of G-CSF–induced signaling in bone marrow MNCs from Gfi1+/+, Gfi1+/−, and Gfi1−/− mice after derivation (time 0, no culture) and after incubation with G-CSF (25 ng/mL) for the indicated time intervals. All results reflect probing of single membranes with specific antibodies. The results are representative of 5 experiments. (D) Quantitative measurement of phopho-Erk1/2 activation by G-CSF (25 ng/mL) in bone marrow MNCs from Gfi1+/+, Gfi1−/−, and Gfi1−/− mice at the indicated time points. The results reflect the mean ± SD band intensity ratios of phospho-Erk1/2/total Erk1/2 (n = 5 mice/group evaluated at each time point). (E) G-CSF–induced Erk1/2 phosphorylation in bone marrow MNCs from Gfi1−/− mice after transduction in vitro with Gfi1-GFP retroviruses, cultured for 48 hours in medium (Iscove DMEM with 10% FCS) supplemented with 10 ng/mL IL-3, 25 ng/mL SCF, 25 ng/mL cKitL, and 5 ng/mL granulocyte-macrophage–CSF, cell sorting the GFP+ and GFP− cells, and starved (1 hour) in culture medium without growth factors. Phospho-Erk1/2 and total Erk was evaluated by fluorescence-activated cell sorting analysis after intracellular staining with specific antibodies. (F) G-CSF–induced signaling evaluated by immunoblotting with specific antibodies. Bone marrow MNCs from Gfi1+/+, Gfi1−/−, and Gfi1−/− mice were incubated with G-CSF (25 ng/mL) for the indicated time intervals. The results reflect reprobing of a single membrane. The results are representative of 3 experiments performed.

Erk activation is selectively defective in G-CSF–stimulated bone marrow cells from Gfi1-null mice. (A) G-CSFR mRNA levels in fresh (left) bone marrow MNCs from untreated Gfi1+/+, Gfi1+/−, and Gfi1−/− mice and after 24-hour incubation in vitro with or without G-CSF (25 ng/mL; right) measured by quantitative real-time PCR. (Left) Gfi1+/+ (n = 3); Gfi1+/− (n = 8); Gfi1−/− (n = 7); the results reflect the means ± SD. (Right) Means (duplicate measurements) of G-CSFR mRNA levels in bone marrow MNCs from individual mice. (B) Proliferation of bone marrow MNCs from Gfi1+/+, Gfi1+/−, and Gfi1−/− mice after 3-day incubation in medium only, with IL-3 (10 ng/mL), or with G-CSF (25 ng/mL). The results reflect the mean 3H thymidine uptake (cpm) of triplicate cultures ± SD and are representative of 3 independent experiments. (C) Immunoblot analysis of G-CSF–induced signaling in bone marrow MNCs from Gfi1+/+, Gfi1+/−, and Gfi1−/− mice after derivation (time 0, no culture) and after incubation with G-CSF (25 ng/mL) for the indicated time intervals. All results reflect probing of single membranes with specific antibodies. The results are representative of 5 experiments. (D) Quantitative measurement of phopho-Erk1/2 activation by G-CSF (25 ng/mL) in bone marrow MNCs from Gfi1+/+, Gfi1−/−, and Gfi1−/− mice at the indicated time points. The results reflect the mean ± SD band intensity ratios of phospho-Erk1/2/total Erk1/2 (n = 5 mice/group evaluated at each time point). (E) G-CSF–induced Erk1/2 phosphorylation in bone marrow MNCs from Gfi1−/− mice after transduction in vitro with Gfi1-GFP retroviruses, cultured for 48 hours in medium (Iscove DMEM with 10% FCS) supplemented with 10 ng/mL IL-3, 25 ng/mL SCF, 25 ng/mL cKitL, and 5 ng/mL granulocyte-macrophage–CSF, cell sorting the GFP+ and GFP− cells, and starved (1 hour) in culture medium without growth factors. Phospho-Erk1/2 and total Erk was evaluated by fluorescence-activated cell sorting analysis after intracellular staining with specific antibodies. (F) G-CSF–induced signaling evaluated by immunoblotting with specific antibodies. Bone marrow MNCs from Gfi1+/+, Gfi1−/−, and Gfi1−/− mice were incubated with G-CSF (25 ng/mL) for the indicated time intervals. The results reflect reprobing of a single membrane. The results are representative of 3 experiments performed.

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