NF-κB activation in LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors in response to GM-CSF. (A) IκBα was measured by intracellular flow cytometry in LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ progenitors at 20 and 40 minutes upon GM-CSF (50 ng/mL) stimulation. (B) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS⁺ and LKS⁻ CD34⁺ FcγR⁺ cells after 3 hours with or without GM-CSF (50 ng/mL) stimulation; scale bar, 2 μm. (C) LKS⁺ 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 NF-κB inhibitor BMS345541 (2 μM) was added 1 hour before and during the 24 hours of GM-CSF stimulation, cells were washed, and differentiated into macrophages with M-CSF for 8 more days. (D-E) TNF-α and IL-6 production was measured by ELISA in supernatants (D), and by intracellular flow cytometry upon LPS stimulation (E). 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 analysis of variance (ANOVA) followed by the Dunnett test for multiple comparisons (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). ab, antibody; AU, arbitrary units; BMS, BMS345541; mφ, macrophage.