Figure 6.

Analysis of expression of Egr2 on a protein level in M1- and M2-like macrophages with knockdown of Egr2 and intracellular cytokine protein level of TNF and IL-6 in IFNγ-activated macrophages with knockdown of Egr2. Bone-marrow-derived macrophages (BMDMs) were transfected with Egr2 siRNA or Control siRNA for 24 h as described in Materials and Methods, and after which the cells were activated with IL-4 or IFNγ for another 24 h-time period as in Figure 5. The cells were washed, stained for surface F4/80, fixed/permeabilized and stained for intracellular Egr2, IL-6, or TNF with fluorescently labeled mAbs and F4/80⁺ cells were analyzed by three-color flow cytometry as described in Materials and Methods. Expression of Egr2 (A,B), IL-6 (C,D) and TNF (E,F) in macrophages transfected with Egr2 siRNA (solid line) vs. Control siRNA (dotted line) are shown on representative histogram graphs (A,C,D). Staining with isotype-matched control mAbs (Control ISO) is shown by shaded histograms. Quantitative analysis (mean fluorescence intensity level for Egr2 and percentage of positive cells for IL-6 and TNF) is shown in (B,D,F). In (B,D,E), mean ± S.E. of six separate culture plate wells is shown (^**p < 0.01; ^***p < 0.001).