Table 1.
Primer sequences.
Table 2.
Genotype and number of newborns from BbEe intercrosses.
Figure 1.
C/EBPβ−/−C/EBPε−/− mice are highly susceptible to fatal infections.
(A) Comparison between wild-type (BBEE) and C/EBPβ−/−C/EBPε−/− (bbee) mice (both at 4 weeks of age). The bbee mice were smaller than BBEE mice and had rougher hair. (B) Body weight of four genotypic subtypes of mice (n = 3 per genotype). Body weights of bbEE and BBee mice were comparable to BBEE mice; but bbee mice weighed less than the other subtypes of mice at 4 weeks after birth (p = 0.05, t-test). (C) Kaplan Meier survival curves of four genotypic subtypes of mice. The bbee mice died 2–3 months after birth due to fatal bacterial infections. (D) Enlarged spleen and abscess formation in the liver in the bbee mice. Spleen size (left panel) was increased in the bbee mice (2 months). Liver (right panel) had visible abscesses (2 months).
Table 3.
Blood cell counts in BBEE, bbEE, BBee, and bbee mice.
Figure 2.
Morphological characterization of neutrophils harvested by lavage from peritoneal cavity.
Morphological characterization of neutrophils harvested by lavage from peritoneal cavity. Wild-type, C/EBPβ−/− (bbEE), C/EBPε−/− (BBee) and C/EBPβ−/−C/EBPε−/− (bbee) mice were injected with 1.5 mL of 4% thioglycollate into their peritoneal cavity. After 18 h, neutrophils were harvested by lavage. Cytopreps of cells were stained with Diff-Quick.
Figure 3.
Characterization of hematopoietic cells in C/EBPβ−/−C/EBPε−/− mice.
(A) Colony assay in methylcellulose. Light-density, mononuclear bone marrow cells (2×104) were cultured in 1% methylcellulose supplemented with either IL-3 (10 ng/mL), IL-6 (10 ng/mL), SCF (20 ng/mL), and EPO (3 U/mL); or GM-CSF (20 ng/mL); or G-CSF (60 ng/mL); or G-CSF (60 ng/mL) and SCF (20 ng/mL). Colonies were scored if they contained ≥50 cells at day 7 of culture. Results represent the mean ± SD of 3 mice. (B) Expression of G-CSF receptor (G-CSFR) in mononuclear bone marrow cells. Expression of G-CSFR mRNA was examined by RT-PCR. β-actin was used as an internal control. (C) Expression of STAT3 in mononuclear bone marrow cells. The cells were stimulated either with or without G-CSF (100 ng/mL) for 30 min. Levels of phospho-STAT3 (pSTAT3) and STAT3 were detected by Western blot analysis.
Figure 4.
Ratio of hematopoietic progenitor cells.
The lineage-negative fraction of bone marrow cells was sorted with FITC-conjugated anti-c-Kit and PE-conjugated anti-Sca-1 antibodies. The hematopoietic progenitor cell population [KSL population (c-Kit+, Sca-1+, and Lineage−; blue dots)] was compared between the wild-type (BBEE), C/EBPβ−/− (bbEE), C/EBPε−/− (BBee) and C/EBPβ−/−C/EBPε−/− (bbee) mice.
Figure 5.
Cluster analysis of microarray data of bone marrow derived macrophages stimulated with LPS and mIFNγ.
Expression data established from activated macrophages revealed a total of 145 genes that were collectively deregulated in the comparison of cells from either wild-type (BBEE), C/EBPβ−/− (bbEE), or C/EBPε−/− (BBee) with C/EBPβ−/−C/EBPε−/− (bbee). Cluster analysis performed with Multi Experiment Viewer (MeV) v4.4.1 indicates 45 of the most deregulated genes out of the collective of 145 genes.
Figure 6.
Validation of mRNA expression of Mrc1, Clec4e, Marco, Socs2, Tnfα, and Cd38 in bone marrow-derived macrophages stimulated with LPS and mIFNγ.
Expression levels of macrophage mannose receptor 1 (Mrc1), C-type lectin domain family 4, member e (Clec4e), macrophage receptor with collagenous structure (Marco), suppressor of cytokine signaling 2 (Socs2), Tnfα and Cd38 mRNA in bone marrow derived macrophages stimulated with LPS and mIFNγ were determined by quantitative RT-PCR. C/EBP-binding site in the promoter region of these genes are also indicated.
Figure 7.
(A, B) Microarray expression analysis established from activated macrophages resulted in 2 pathways which were significantly (p<0.001) involved in the comparison of wild-type (BBEE), C/EBPβ−/− (bbEE), and/or C/EBPε−/− (BBee) with C/EBPβ−/−C/EBPε−/− (bbee). They both represent inflammatory response pathways involving pro-inflammatory cytokines and chemokines, as well as Toll-like receptor signaling molecules. Solid lines denote direct interactions, while dotted lines represent indirect interactions of genes/proteins. Green: downregulated (≤2 fold); Red: upregulated (≥2 fold) molecules.
Figure 8.
Chromatin immunoprecipitation (ChIP) assay in bone marrow derived macrophages stimulated with LPS and mIFNγ.
Bone marrow derived macrophages stimulated with LPS and mIFNγ were subjected to ChIP assay by using anti-C/EBPβ, anti-C/EBPε or normal IgG antibodies. C/EBP-binding region of the Marco (A) and Clec4e (B) genes were amplified by specific primers.