Table 1.
Primers used for Real-time PCR analysis.
Fig 1.
Loss of IKKβ results in increase granulocytes in vivo and ex vivo.
A) Equal number of Lin- bone marrow cells from wild-type (WT), IKKβΔ/Δ, or p65Δ/Δ mice were subjected to immunoblotting with the indicated antibodies. Representative gels with relative band intensity values are shown. B) Genomic DNA was extracted from the tail or bone marrow of mice with the indicated genotypes before or after pIpC injection and subjected to PCR analysis using primer sets that anneal to the excised fragment. C) Marrow cells from mice of the indicated genotypes were stained for Mac-1 and Gr-1 (top) and analyzed by FACS. Mac-1+ cells (middle) were stained for Gr-1 and CD115 (bottom). D) Peripheral blood leukocytesfrom 26 weeks old mice were subjected to Wright-Giemsa staining and representative morphology is shown. E) Equivalent numbers of mononuclear marrow cells from wild type (WT), IKKβΔ/Δ, or p65Δ/Δ mice were plated in methylcellulose with IL-3, IL-6, and SCF, and CFU-G, CFU-M, and CFU-GM colonies were enumerated 8 days later. The average number of colonies from 4 experiments is shown. F) Bone marrow cells were cultured with the indicated combinations of 20 ng/mL murine TNFα for 30 min, with or without 4 hrs pre-incubation with Bay 65–1942 at the indicted dose. Cell lysates were subjected to Western blotting with the indicated antibodies and the numbers below the blots indicate the relative band densities. G) Bone marrow cells were plated in methylcellulose with IL-3, IL-6, and SCF in the presence or absence of 0.5 μM Bay 65–1942 and colonies were enumerated 8 days later. The average number of colonies from 3 experiments is shown (right).
Table 2.
Complete blood counts and spleen weights 6 wks after poly(I:C) injections.
Fig 2.
Deletion of IKKβ is associated with increased number of myeloid progenitors and hematopoietic stem cells.
A) Bone marrow cells were harvested from the tibias of wild type or IKKβΔ/Δ mice, and the average number of cells is presented (n = 4). B) Bone marrow cells from wild-type (WT), IKKβΔ/Δ or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32 and CD135. The indicated progenitor populations were identified and representative plots and the percent ± SEM of each population relative to total Lin- marrow cells are shown (n = 4). C) The actual number of cells for the indicated progenitor populations per hind leg was calculated and averages from at least 4 experiments are shown. D) The average absolute number of multipotential progenitors (MPP) and long and short term hematopoietic stem cells (LT- and ST-HSC) per hind leg are shown (n = 4). E) CD45.1 mice were lethally irradiated and then intravenously injected with 1E6 CD45.2 marrow cells from wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx-1-Cre mice. Eight wks after transplant mice were intraperitoneally injected with poly(I:C) for 7 doses starting four weeks after transplantation. Marrow harvested 6 week later and the average absolute number of the indicated progenitor and stem cell populations per hind leg are shown (n = 3).
Fig 3.
IKKβ deletion is associated with reduced apoptosis and altered cell cycle distribution.
A) Bone marrow cells from wild type, IKKβΔ/Δ, or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32, and Annexin V. The average proportion of cells positive for Annexin V for each of the indicated progenitor populations is shown (n = 3). B) The indicated populations from wild type, IKKβΔ/Δ, or p65Δ/Δ mice were sorted, stained with propidium iodide and DNA content was analyzed by flow cytometry. The mean distribution +/- SEM of cells in G1, S, or G2/M from four experiments are presented as well as C) the ratio of the proportion of cells in G1 over S phase from four experiments are presented (n = 4). *-denotes p<0.01.
Fig 4.
IKKβ deletion is associated with impaired erythropoiesis.
A) Bone marrow cells from wild-type (WT) or IKKβΔ/Δ mice were stained for CD11b, CD45, Ter119 and CD71. CD11b-;CD45- cells were gated and the proportion of proerythroblasts, basophilic, polychromatic, and orthochromatic erythroblasts in nucleated bone marrow cells is shown on representative FACS plots. B) The average number of cells of each of erythroid subpopulation per hind leg is shown (n = 4). C) Equal number of marrow cells from wild type, IKKβΔ/Δ or p65Δ/Δ mice were plated in methylcellulose under conditions promoting erythroid maturation, and BFU-Es were enumerated after 8 days. Average number of colonies from 3 experiments is shown. D) Wild type bone marrow cells were plated in methylcellulose in the presence or absence of 1 μM Bay 65–1942, and BFU-Es were enumerated 8 days later (n = 3). Hematocrit was assessed in wild type or IKKβΔ/Δ mice after induction of hemolysis with E) PHZ 200 mg/kg or F) challenge with 250 mg/kg 5-FU. G) Neutrophil recovery after 5-FU. *-denotes p<0.01. **denotes p<0.001.
Fig 5.
Deletion of IKKβ is associated with replating and repopulation advantage.
A) Equal number of wild type, IKKβΔ/Δ or p65Δ/Δ bone marrow cells were plated and myeloid CFUs were enumerated and replated every 7 days. The average number of colonies at the indicated replating generation is shown. B, C) Lethally irradiated CD45.1 mice were injected with 5E5 CD45.2 marrow cells from wild type or IKKβΔ/Δ along with 5E5 CD45.1 marrow cells. Engraftment of bone marrow was assessed using FACS analysis for CD45.1 and CD45.2. Representative marrow FACS plots and the average proportion of CD45.2 engraftment in the marrow 20 weeks after transplant is shown (n = 5).
Fig 6.
Deletion of IKKβ is associated with altered expression of hematopoietic transcription factors.
Total cellular RNA was isolated from Lin- marrow cells of mice with the indicated genotypes and the expression of A) myeloid differentiation associated genes, B) hematopoietic transcription regulators C) transcription factors regulating erythropoiesis, and D) anti-apoptotic Bcl2 family members was analyzed using qRT-PCR. Average relative expression from 3–4 independent experiments is shown. E) LSK cells were isolated by FACS from wild type or IKKβΔ/Δ mice, RNA was extracted and subjected to qRT-PCR. *-denote p<0.01. **denotes p<0.001. F) Cell lysates were obtained from wild-type (WT) or IKKβΔ/Δ Lin- bone marrow cells and subjected to Western blot analysis with the indicated antibodies. Representative gels with relative band intensity values are shown.
Table 3.
Effects of IKKβ or p65 deletion on hematopoiesis.