Figure 5
Absence of C/EBPβ dramatically decreases microglial NOS2 protein expression and NO production in activated mixed glial cultures. A. NOS2 protein levels in total protein extracts from mixed glial cultures were analyzed by western blot, followed by densitometry. Data is expressed as NOS2 versus β-actin band intensities. Cultures were treated for 16 h with LPS, LPS+IFNγ or vehicle. In C/EBPβ+/+ cultures (white bars) NOS2 protein levels were detected after LPS treatment, but LPS+IFNγ induced a clear upregulation, in agreement with mRNA expression levels. In C/EBPβ-/- mixed glial cultures (black bars), NOS2 protein levels decreased in LPS and LPS+IFNγ compared to C/EBPβ+/+ cultures. Two-way ANOVA, followed by Bonferroni's test was applied. ***p < 0.001 compared with C/EBPβ+/+ condition. ###p < 0.001 compared with respective control condition. (n = 5). A representative western blot is shown in B. C. NO production is decreased in activated C/EBPβ-/- glial cultures. NO levels were measured by colorimetric analysis 48 h after treatments and normalized per cell number. Values are reported as micromolar concentration ×106 cells. NO levels in C/EBPβ+/+ (white bars) cultures were upregulated after LPS and LPS+IFNγ treatments compared to controls. In C/EBPβ-/- glial cultures (black bars), NO production is reduced in LPS+IFNγ treatment compared to wild-type NO levels. Two-way ANOVA, followed by Bonferroni's test was applied. ***p < 0.001 compared to C/EBPβ+/+ condition. ###p < 0.001 compared to respective control condition. %%%p < 0.001 compared to respective LPS condition. (n = 7-9). D. NOS2 is expressed by activated microglia. C/EBPβ+/+ and C/EBPβ-/- secondary mixed glial cultures were immunostained for CD11b (green) and NOS2 (red), and stained for Hoechst 33258 (blue) after 16 h of LPS+IFNγ treatment. The C/EBPβ+/+ merged image shows colocalization of NOS2-positive cells and CD11b-positive cells. Bar = 50 μm.