Signaling pathways involved in the accumulation of β-catenin in LKS⁻ CD34⁺ FcγR⁺ progenitors in response to GM-CSF. (A) β-catenin was measured by intracellular flow cytometry in LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors at 24 hours upon GM-CSF (50 ng/mL) stimulation. (B) Microphotographs of β-catenin location by immunofluorescence in LKS⁻ CD34⁺ FcγR⁺ cells after 24 hours of GM-CSF (50 ng/mL) stimulation; scale bar, 2 μm. (C) β-catenin was measured by intracellular flow cytometry in LKS⁻ CD34⁺ FcγR⁺ progenitors at 24 hours upon GM-CSF (50 ng/mL) stimulation with or without the indicated inhibitors (UO216 10 μM, ZSTK474 1 μM, STAT5i 10 μM and Rapamycin 40 ng/mL) 1 hour before and during stimulation. (D) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS⁻ CD34⁺ FcγR⁺ cells after 3 hours of GM-CSF (50 ng/mL) stimulation with or without the indicated inhibitors 1 hour before and during stimulation; scale bar, 2 μm. (E) LKS⁻ CD34⁺ FcγR⁺ cells were isolated from the bone marrow of C57BL/6 mice, cultured with M-CSF (50 ng/mL) + SCF (20 ng/mL) and stimulated with or without GM-CSF (50 ng/mL) for 24 hours, the indicated inhibitors or dimethyl sulfoxide control solvent were added 1 hour before and during the 24 hours of GM-CSF stimulation, cells were washed, and differentiated into macrophages with M-CSF for 6 more days. TNF-α production upon LPS stimulation was measured by ELISA in supernatants and by intracellular flow cytometry. For the ELISA measurements triplicate samples were analyzed and expressed as means + SD and for intracellular cytokine measurements, percentage of cytokine-producing cells and MFI are indicated in the heatmaps, and bar graphs for MFIs and box plots for cell percentages are expressed as means + SD. Data presented are 1 representative experiment of at least 3 independent experiments. Statistical significance was assessed by the Student t test and the 1-way ANOVA followed by the Dunnett test for multiple comparisons (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). AU, arbitrary units; i.c., isotype control; Rapa., rapamycin; unstim. or Ø, unstimulated; UO, UO216; ZSTK, ZSTK474.