Signaling pathways activated in LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors in response to GM-CSF. (A) Phosphorylated STAT5 (pSTAT5), ERK (pERK), and Akt (pAkt) were measured by intracellular flow cytometry at 10 and 30 minutes, and S6 (pS6) at 4 hours, in GM-CSF–stimulated (50 ng/mL) LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors. The expression of CD116 and CD131 was measured on the surface of LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors and Ly6C^hi monocytes (Ly6G⁻ CD11b⁺ CD115⁺ Ly6C^hi) (B); and on the surface of LT-HSC, ST-HSC, MPP2, MPP3 and MPP4 progenitors (C) by flow cytometry. MFI measurements are means + SD of 3 independent experiments and histograms are from 1 representative experiment. LT-HSC, ST-HSC, MPP2, and MPP3 progenitors isolated from the bone marrow of mice were cultured in vitro with M-CSF (50 ng/mL) + SCF (20 ng/mL) and stimulated with or without GM-CSF (50 ng/mL) for 24 hours, washed and differentiated into macrophages with M-CSF for 8 days (MPP2 and MPP3) or 10 days (LT- and ST-HSCs); TNF-α production was measured by intracellular flow cytometry upon LPS stimulation (D) and by ELISA in supernatants upon LPS stimulation (E). For the ELISA measurements triplicate samples were analyzed and expressed as means + SD and for intracellular cytokine measurements MFI is indicated in the histograms; data presented are 1 representative experiment of at least 3 independent experiments. Statistical significance was assessed by the Student t test (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). i.c., isotype control; unstim., unstimulated.