Figure 6.
Nuclear NF-κB levels in GM-CSF–stimulated LKS+ cells upon GSK3 inhibition. (A) β-catenin was measured by intracellular flow cytometry in LKS+ progenitors at 24 hours upon GM-CSF (50 ng/mL) stimulation with or without LiCl (10 mM) 1 hour before and during stimulation. (B) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS+ cells after 3 hours of GM-CSF (50 ng/mL) stimulation with or without LiCl 1 hour before and during stimulation; scale bar, 2 μm. (C-D) 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 GSK3 inhibitor LiCl (10 mM) 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. TNF-α and IL-6 production by macrophages upon LPS stimulation was measured by ELISA in supernatants (C), and TNF-α by intracellular flow cytometry (D). (E) IκBα was measured by intracellular flow cytometry in LKS+ progenitors at 20 minutes upon TNF-α (100 ng/mL) stimulation. (F) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS+ cells after 3 hours with or without TNF-α (100 ng/mL) stimulation; scale bar, 2 μm. (G-H) LKS+ cells were treated as in panels C-D, but using TNF-α (100 ng/mL) for stimulation instead of GM-CSF, and TNF-α and IL-6 production by macrophages upon LPS stimulation was measured by ELISA in supernatants (G), and TNF-α by intracellular flow cytometry (H). 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 is 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 and ∗∗∗P < .001). AU, arbitrary units; i.c., isotype control; mφ, macrophage.

Nuclear NF-κB levels in GM-CSF–stimulated LKS+ cells upon GSK3 inhibition. (A) β-catenin was measured by intracellular flow cytometry in LKS+ progenitors at 24 hours upon GM-CSF (50 ng/mL) stimulation with or without LiCl (10 mM) 1 hour before and during stimulation. (B) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS+ cells after 3 hours of GM-CSF (50 ng/mL) stimulation with or without LiCl 1 hour before and during stimulation; scale bar, 2 μm. (C-D) 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 GSK3 inhibitor LiCl (10 mM) 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. TNF-α and IL-6 production by macrophages upon LPS stimulation was measured by ELISA in supernatants (C), and TNF-α by intracellular flow cytometry (D). (E) IκBα was measured by intracellular flow cytometry in LKS+ progenitors at 20 minutes upon TNF-α (100 ng/mL) stimulation. (F) Microphotographs of p65 location (left) and nuclear p65 mean fluorescence quantification (right) by immunofluorescence in LKS+ cells after 3 hours with or without TNF-α (100 ng/mL) stimulation; scale bar, 2 μm. (G-H) LKS+ cells were treated as in panels C-D, but using TNF-α (100 ng/mL) for stimulation instead of GM-CSF, and TNF-α and IL-6 production by macrophages upon LPS stimulation was measured by ELISA in supernatants (G), and TNF-α by intracellular flow cytometry (H). 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 is 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 and ∗∗∗P < .001). AU, arbitrary units; i.c., isotype control; mφ, macrophage.

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