Myeloid lineage analysis of bone marrow cells. Bone marrow cells were isolated from wild-type (WT; n = 4) and C/EBPβ-deficient (KO; n = 4) adult mice, stained with fluorescent-labeled antibodies against myeloid lineage-specific antigens (Mac1/CD11b, CD14, and Gr-1), and analyzed by flow cytometry. Results represent the mean plus or minus SD of 4 mice in each cohort.

Apoptosis assay of neutrophils. Neutrophils of wild-type and C/EBPβ-deficient mice were harvested from peritoneal cavity and cultured for 0, 2, 6, and 18 hours with RPMI 1640 and 10% FBS alone (A) or with LPS (100 ng/mL) (B) or GM-CSF (10 ng/mL) (C). Apoptosis was measured by TUNEL assay. Results represent the mean plus or minus SD of 4 mice in each cohort.

Differential cytokine gene expression in primary macrophages and neutrophils. Macrophages (A) and neutrophils (B) from wild-type and C/EBPβ-deficient mice were harvested from peritoneal cavity and cultured either with (+) or without (−) LPS (100 ng/mL, 4 hours). Total RNAs were isolated, and expression of cytokine genes was quantified by real-time RT-PCR as a ratio of these transcripts/18S transcripts, and wild-type LPS-untreated samples are calculated as 1. ■ and □ show wild-type and C/EBPβ-deficient mice, respectively. Results represent the mean plus or minus SD.

Neutrophil counts in mice after 5-FU treatment. 5-FU (150 mg/kg) was given intraperitoneally to the wild-type and C/EBPβ-deficient mice. Blood samples were taken from the ocular sinus at 0, 1, 4, 7, 10, and 14 days, and neutrophils were counted. Black and broken lines show their mean plus or minus SD results from wild-type and C/EBPβ-deficient mice, respectively.

Analysis of bone marrow cells. Bone marrow cells were isolated from wild-type (n = 4) and C/EBPβ-deficient (n = 4) adult mice. (A) Bone marrow cells (2 × 10⁴) were cultured in 1% methylcellulose supplemented with 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) and SCF (20 ng/mL); or G-CSF (60 ng/mL) alone. Colonies were scored if they contained more than 50 cells at day 7. Results in panels A and B represent the mean plus or minus SD of 4 mice in each cohort (*P < .001; **P < .005). (B) Bone marrow cells (2 × 10⁵) were cultured in either 1% methylcellulose or MegaCult-C media (CFU-Meg), supplemented with either EPO (3 U/mL); or EPO (3 U/mL) and SCF (20 ng/mL); or TPO (50 ng/mL), IL-3 (10 ng/mL), IL-6 (20 ng/mL), and IL-11 (10 ng/mL). BFU-E (EPO and SCF) and CFU-Meg (TPO, IL-3, IL-6, and IL-11) were counted on day 7 of culture; CFU-E (EPO alone) was enumerated on day 2. Black and white bars show wild-type and C/EBPβ-deficient mice, respectively. (C) Detection of phospho-STAT3 in bone marrow cells. Bone marrow cells were isolated from C/EBPβ heterozygous mutant and C/EBPβ-deficient adult mice, and the cells were stimulated either with or without G-CSF (100 ng/mL) for 30 minutes. Signals of phospho-STAT3 (pSTAT3) and STAT3 were detected by Western blot analysis.