Figure 1.
TAK1, TAB1 and TAB2 in bone marrow cells.
(A) Expression of TAK1, TAB1 and TAB2 proteins. Whole cell extracts of the BMN cells (BM), splenocytes (Spleen) and thymocytes (Thymus) were analyzed by SDS-PAGE and Western blotted (WB) using the indicated antibodies. The amount of β-actin is shown as a loading control. (B) Expression of Tak1, Tab1 and Tab2 mRNA. Total RNA was isolated from BMN cells (BM), splenocytes (Spleen) and thymocytes (Thymus), and analyzed by qPCR. Expression level of each gene was normalized to that of Actb and shown as the relative value to BM. Data are presented as mean ± S.D. of three independent experiments. (C) Expression of Tak1, Tab1 and Tab2 mRNA. The cells in the LT-HSC (CD34− Flt3− LSK), ST-HSC (CD34+ Flt3− LSK), MPP (CD34+ Flt3+ LSK), MP (Lineage− c-Kit+ Sca-1−) or Lineage+ fractions from wild type mouse bone marrow were sorted by FACS, and total RNA was prepared. The relative amounts of Tak1, Tab1, Tab2 and Actb mRNA were determined by qPCR. Expression level of each gene was normalized to that of Actb and shown as the relative value to LT-HSC. Data are presented as mean ± S.D. of four independent experiments.
Figure 2.
Competitive reconstitution assay.
(A) Schematic representation of competitive transplantation. 2×105 BMN cells from Cre-alone, Tak1iKO or Tab1Tab2iDKO mice (CD45.2+) were transplanted into lethally irradiated recipients (CD45.1) together with 2×105 competitor wild type BMN cells (CD45.1+). At six weeks post-transplantation (designated as Week 0), the chimerism of myeloid, T and B cell populations in the recipients’ peripheral blood (PB) was analyzed, then the recipients were i.p. injected with tamoxifen at 160 mg/kg body weight for three consecutive days. (B) The chimerism of PB cell populations was monitored every three weeks, and shown as mean ± S.D. (*p<0.05). [Cre-alone (solid line, circles, n = 3), Tak1iKO (dashed line, squares, n = 3) or Tab1Tab2iDKO (dashed line, triangles, n = 4)]. (C) Representative flow cytometry data of blood cell chimerism. PB cells were collected 15 weeks after tamoxifen injection. Myeloid cells (CD11b+ or Gr-1+), B cells (B220+) and T cells (CD3ε+) in PB mononuclear cells were analyzed for the expression of CD45.1 or CD45.2. (D) Competitive reconstitution assay for Tab2iKO and Tak1iKO mice. Donor-derived chimerism of PBs was analyzed. The percentage of Tab2iKO or Tak1iKO PB myeloid, T and B cells (CD45.2+) in total PB myeloid, T and B cells (CD45.1+ and CD45.2+) before (Week 0, open bars) and 20 weeks or 21 weeks, respectively, after tamoxifen injection is shown (gray bars). Data are presented as mean ± S.D. (*p<0.05, n.s. not significant, n = 3).
Figure 3.
In vitro expansion of LSK population.
(A) The mice with indicated genotypes were i.p. injected with tamoxifen (160 mg/kg) for three consecutive days and sacrificed at day 4. The total LSK, MP and CLP cell numbers in the femurs and tibias from each mouse were determined. Data are presented as mean ± S.D. (n.s. not significant, n = 3) (B) Whole BMN cells isolated at day 4 described in (A) were cultured in STIFA medium. At the time of plating, 2.5×106 cells/well, 2×106 cells/well and 1×106 cells/well were plated for 3-, 6- and 9-day culture, respectively. Cells were harvested and analyzed by flow cytometry for lineage, Sca-1 and c-Kit surface markers. The absolute number of the LSK population in the harvested cells is shown as per 1×106 initial BMN cells. Data are presented as mean ± S.D. (*p<0.05, n = 3) (C, D) Annexin V-binding assay of LSK (C) and lineage positive cells (D) after nine days of cell expansion in STIFA medium. Data are presented as mean ± S.D. (*p<0.05, n = 3).
Figure 4.
(A) The chimerism of LT-HSC, ST-HSC/MPP and MP in the recipient mice BM in competitive transplantation assay. The recipient mice (CD45.1+) were lethally irradiated and transplanted with a mixture of 1×106 test (Tak1Het and Tak1iKO, CD45.2+) and 2×105 competitor wild type (CD45.1+) BMN cells. Three weeks after tamoxifen injection, these recipients were analyzed for the chimerism of LT-HSC (CD34(–), LSK), ST-LSK/MPP (CD34(+) LSK) and MP (LK). Gating strategy and one result for each of the Tak1Het and Tak1iKO test BMN transplanted animals are shown. (B) Gating strategy for SLAM-LSK and one result for each of the Tak1Het and Tak1iKO test BMN transplanted animals are shown. (C) The chimerism of LT-HSC (CD34(–), LSK), ST-LSK/MPP (CD34(+) LSK), MP (LK) (n = 4) and SLAM-LSK (n = 3) in the recipient mice BM. The chimerism in each recipient is plotted, and the bars represent the average. * p<0.05 [Tak1Het versus Tak1iKO or Tab1Tab2iDKO].
Figure 5.
Ablation of TNF signaling partially restores the reconstitution potential of Tak1-deficient HSCs.
(A) Competitive reconstitution assay. 2×105 BMN cells from control Tak1Het Tnfr1−/− (n = 3) or Tak1iKO Tnfr1−/− mice (n = 4) (CD45.2+) were transplanted into lethally irradiated recipients (CD45.1+) together with 2×105 competitor wild type BMN cells (CD45.1+). At six weeks post transplantation (designated as Week 0), the chimerism of myeloid, T and B cells in the recipients’ PB was analyzed, and then the recipients were i.p. injected with tamoxifen at 160 mg/kg body weight for three consecutive days. The chimerism of PB cells was monitored every three weeks, and is shown as the mean ± S.D. (*p<0.05) (B) Competitive reconstitution assay of Cre-alone (n = 3) and Tak1iKO (n = 3) was also performed as described above, and compared with Tak1Het Tnfr1−/− (n = 3) and Tak1iKO Tnfr1−/− (n = 4) at 18 weeks post-tamoxifen injection. The table shown below the graphs indicates statistical significance for the indicated comparisons. P values of less than 0.05 are highlighted. (C) Expression of TAK1 and TAB1 proteins in the donor-derived splenocytes. Whole spleen cells from control Tak1Het and two independent Tak1iKO Tnfr1−/− transplanted mice (#1 and #2) at 15 weeks post-tamoxifen injection were sorted into the CD45.1+ or CD45.2+ population. Total cell lysates from the sorted splenocytes from control Tak1Het, and Tak1iKO Tnfr1−/− #1 and #2 were analyzed by SDS-PAGE and Western blotted with anti-TAK1, TAB1 or anti-β-actin antibodies. The positions of molecular weight markers are shown on the right. The arrows indicate the bands corresponding to endogenous TAK1 and TAB1, and truncated TAK1 (TAK1Δ) resulting from Cre-mediated recombination.
Figure 6.
Impaired LT-HSC function by Tak1 deficiency cannot be rescued by ablation of TNF signaling.
(A) Gating strategy for LT-HSC, ST-HSC/MPP and MP in the competitive reconstitution assay. (B) Representative results of chimerism analysis of No-Cre, Tak1iKO, Tak1iKO Tnfr1−/−, and Tak1Het Tnfr1−/− test donor transplanted mice are shown. (C) The chimerism of LT-HSC, ST-HSC/MPP and MP in the recipient mouse BM. Lethally irradiated recipient mice (CD45.1+) were transplanted with a mixture of 5×105 test (controls, No-Cre and Tak1Het Tnfr1−/−; and Tak1 deficient, Tak1iKO and Tak1iKO Tnfr1−/−, CD45.2+) and 5×105 competitor wild type (CD45.1+) BMN cells. The recipients were i.p. injected with tamoxifen at 160 mg/kg body weight for three consecutive days starting at six weeks post transplantation. Twenty-two weeks or more after tamoxifen injection, recipients were analyzed for the chimerism of LT-HSC (CD34−, LSK), ST-HSC/MPP (CD34+, LSK) and MP (LK) in BMN cells. The chimerism in each recipient is plotted, and the bars represent the average. The table shown below the graphs indicates statistical significance for the indicated comparisons. P values of less than 0.05 are highlighted. Mice were i.p. injected with tamoxifen at 160 mg/kg body for three consecutive days (Tak1iKO, Tab1iKO, Tab2iKO and Tab1Tab2iDKO) or untreated (Tak1FF, Tak1FF Cre or Tab1Tab2FF) and sacrificed on day 4. Genomic DNA isolated from BMN cells was analyzed by qPCR using primers designed to detect a portion of the genome flanked by two loxP sites to determine the relative copy number of intact Tak1, Tab1 or Tab2 genome. Data are presented as mean ± S.D. of three independent experiments. Mice with the indicated genotype were i.p. injected with tamoxifen (160 mg/kg) for three consecutive days, and splenocytes were collected at Day 14 (left panels) or Day 4 (right panels). Whole cell extracts were prepared and analyzed by Western blot using the indicated antibodies. Asterisks indicate non-specific bands. Mice with the indicated genotype were i.p. injected with tamoxifen (160 mg/kg body weight) for three consecutive days, and BMN cells were collected at Day 4. (A) The number of total BMN cells of femurs and tibias were counted on hemacytometer. The cell number in MP or LSK population was determined by FACS analysis. Data are presented as mean ± S.D. (n = 3) (*p<0.05) (B) Representative FACS plots for Sca-1 vs c-Kit in lineage-negative population of BMN cells are shown. The gates for MP and LSK and percentages of each population are indicated. (A) 2×105 BMN cells from Tab1FF, Tab1iKO, Tab2FF, or Tab2iKO mice (CD45.2+) were transplanted into lethally irradiated recipients (CD45.1+) together with 2×105 competitor wild type BMN cells (CD45.1+). At six weeks post transplantation, the chimerism of myeloid, T and B cells in the recipients’ PB was analyzed, then the recipients were i.p. injected with tamoxifen (160 mg/kg body weight) for three consecutive days. The chimerism of PB cells was monitored every three weeks. In each experiment, a CD45.1/CD45.2 mixture of BMN cells was transplanted into two recipients, and the average of their blood cell chimerism is shown. (B) Splenocytes from control No-Cre or Tab2iKO transplanted mice (#1 and #2) at 21 weeks post-tamoxifen injection were sorted into the CD45.1+ or CD45.2+ population. Total cell lysates from the sorted splenocytes were analyzed by SDS-PAGE and Western blotted with indicated antibodies. The positions of molecular weight markers are shown on the right. The asterisks indicate the non-specific bands. Mice with the indicated genotype (the same animals used for the in vitro LSK expansion assay in Figure 3) were i.p. injected with tamoxifen (160 mg/kg body weight) for three consecutive days, and BMN cells were collected at Day 4. BMN cells were analyzed by FACS to determine the number of B cell (B220+), T cell (CD3ε+) and granulocyte (CD11b+ and Gr-1+) per femurs and tibias. Data are presented as mean ± S.D (n = 3).