Fig 2: Analysis of reciprocal regulation of CEBPß and Egr2 in M1 and M2 macrophages. (A) Effect of inhibition of Egr2 on CEBPß expression in M0, M2, and M1 macrophages. Bone-marrow-derived macrophages (BMDMs) were transfected with siRNA cocktail for Egr2 [Egr2(si)] or control siRNA [C(si)] for 24 h as described in Materials and Methods, and after which the cells were used as unstimulated [C(si) and Egr2(si)] or activated with IL-4 [C(si) + IL4 and Egr2(si) + IL4] or IFN? [C(si)+IFN and Egr2(si)+IFN] for another 24 h-time period as in Figure 3. The cells were washed, mRNA was isolated and the expressions of CEBPß was analyzed by real-time PCR as described in Materials and Methods. (B) Effect of overexpression of Egr2 on CEBPß expression in M1 macrophages. Macrophage cell line RAW264.7 was transfected with pMIG expression vector plasmid or empty plasmid vector [V(C) or vector with Egr2 (V(Egr2)] for 24 h and after which the cells were used as unstimulated [V(C) and V(Egr2)] or activated with LPS for another 24 h-time period [V(C)+LPS and V(Egr2)+LPS] as in Figure 8. The cells were washed, mRNA was isolated and the expressions of CEBPß were analyzed by real-time PCR as described in Materials and Methods. (C) Effect of inhibition of CEBPß on Egr2 expression in unstimulated (M0), M2, and M1 macrophages. BMDMs were transfected with siRNA cocktail for CEBPa [CEBPA(si)], CEBPß [CEBPB(si)], or control siRNA [C(si)] for 24 h as described in Materials and Methods. After which, the cells were used as unstimulated [C(si), CEBPA(si), and CEBPB(si)] or activated with IL-4 [C(si)+IL4, CEBPA(si)+IL4, and CEBPB(si)+IL4] or IFN? [C(si)+IFN, CEBPA(si)+IFN and CEBPB(si)+IFN] for another 24 h-time period as in Figure 3. The cells were washed, mRNA was isolated and the expressions of Egr2 was analyzed by real-time PCR as described in Materials and Methods. (D) Kinetics of expression of CEBPß on mRNA level in M2 (IL-4) and M1 (IFN?/LPS) macrophages. BMDMs were stimulated with IL-4 or IFN?/LPS and the levels of CEBPß expression was determined by real-time RT PCR immediately (0 h), 8, and 24 h after activation similarly as for Figure 2. Mean ± S.D. of triplicate is shown (*p < 0.05; **p < 0.01; ****p < 0.0001 when compared to untreated (0 h) cells). (E) Expression of CEBPß on protein level in M2 (IL-4) and M1 (IFN?/LPS) macrophages. BMDMs were used unstimulated (Cont) or stimulated with IL-4, or IFN? with LPS for 24 h as for Figure 3 and the level of expression of CEBPß was analyzed by western blot as described in Materials and Methods. A representative blot (upper image) and quantitative analysis (bottom graph) are shown here. The whole blots are shown in Figure S7. (F) Model of reciprocal regulation of CEBPß and Egr2 in M1 and M2 macrophages. In both M1 and M2 macrophages Egr1 upregulate CEBPß, which in turn induce expression of M1 or M2 markers, respectively. In M1 macrophages, high level of CEBPß inhibits Egr2 leading to non-responsiveness of these cells to further simulation. In (A–C,E), mean ± S.E. of 4–6 separate culture plate wells is shown (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; *****p < 0.00001).