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stem1577-sup-0001-suppfig1.jpg706KFigure S1. Determination of the optimal number of donor cells necessary for the survival of recipient mice after irradiation. After limiting dilution, different numbers of BM MNCs (2.5 × 105, 5 × 105, 1 × 106, or 2 × 106) were transplanted into recipient mice that had been irradiated with 12 Gy. The survival rate was monitored for 30 days after transplantation. Saline was administrated orally to mice for five consecutive days as a control (arrows). Data from two independent experiments are shown (n = 5 each experiment).
stem1577-sup-0002-suppfig2.jpg2781KFigure S2. The specificity of the antibodies used for the immunohistochemical analyses. Representative images of tPA immunostaining in the BM of the tPA KO mice and of PAI-1 immunostaining in the BM of the PAI-1 KO mice. Two days after irradiation with 9 Gy, a femur was removed, paraffin sections were prepared and immunostaining was performed as described in the Materials and Methods section. The immunostaining of tPA and PAI-1 was undetectable in the tPA-KO and PAI-1 KO mice, respectively, confirming the specificity of these antibodies (please note the positive immunostaining for tPA and PAI-1 in the WT mice, Fig. 1B). The lower images show that the sections were stained with isotype-matched control antibodies.
stem1577-sup-0003-suppfig3.jpg2127KFigure S3. Representative images of immunofluorescent studies of the BM from untreated [lsqb]rad (-)[rsqb] and irradiated [lsqb]rad (+)[rsqb] mice. Two days after irradiation with 9 Gy, a femur was removed, and sections were prepared. Double immunostaining for (A) CD31 (red) and tPA (green), (B) PAI-1 (green) and col (I) ?1 (red) or (C) PAI-1 (green) and osteocalcin (red). Nuclei were counterstained with DAPI (blue). Arrowheads indicate positive cells. The bottom raw images show that the sections obtained from the irradiated mice were not stained with the first antibodies (staining with the second antibodies only was observed).
stem1577-sup-0004-suppfig4.jpg927KFigure S4. The expression levels of the genes and proteins of fibrinolytic factors and PAI-1 in cultured BM cells after irradiation. The mRNA levels of tPA, Plg and PAI-1 24 hours after 10 Gy irradiation in the primary culture of BM stromal cells (A) or in the BM stromal cell line, HESS5 (B), were evaluated by quantitative RT-PCR using TaqMan probes (Gene ID for tPA, Plg, and PAI-1 were Mm00476931_m1, Mm00447087_m1, and Mm00435860_m1, respectively). (C): The concentrations of the tPA, plasmin and PAI-1 proteins in the culture medium of the BM stromal cell line were evaluated by ELISA. The data from three independent experiments are shown as the means ? SD (n = 6 for each condition). *p [lt] .05.
stem1577-sup-0005-suppfig5.jpg2469KFigure S5. The results of the FACS analysis of donor-derived HSCs in the reconstituted recipient BM. The WT or PAI-1 KO mice with a Ly5.2+ background were irradiated with 9 Gy and subsequently transplanted with 2.5 × 106 BM MNCs from the Ly5.1+ WT mice. Representative FACS profiles of donor-derived Lin-SLAM cells at one week (A), and of donor-derived CD34-LSK cells at one (B) or three (C) weeks after transplantation. The numbers (%) in some FACS diagrams indicate the percentage of the gated cells in the diagram. The numbers (%) in parentheses indicate the percentages of donor-derived BM MNCs. The Ly5.1+donor-derived cells constituted more than 90% of the reconstituted hematopoietic cells in the WT, as well as PAI-1 KO, mice (n = 18 each). The experiments were repeated three times, and the representative results from one experiment are shown.
stem1577-sup-0006-suppfig6.jpg819KFigure S6. The effects of the PAI-1 inhibitor on the proportion of Mac-1+/Gr-1+ myeloid cells and B220+ lymphoid cells among Ly5.1+ donor cells at one and three weeks after transplantation. The Ly5.2+ recipient mice were irradiated, transplanted with Ly5.1+ donor cells and given a PAI-1 inhibitor (100 mg/kg, p.o.) daily for five consecutive days. Data from four independent experiments (n = 12) are shown as the means ? SD.
stem1577-sup-0008-suppfig7.jpg317KFigure S7. The effects of tPA and the PAI-1 inhibitor on the apoptosis of transplanted LSK cells. The Ly5.1+ BM MNCs were transplanted into the 9 Gy irradiated Ly5.2+ mice. Either tPA (10 mg/kg, i.p.) or the PAI-1 inhibitor (100 mg/kg, p.o.) was administered daily for five consecutive days. One week after transplantation, BM MNCs were collected, and the proportion of Annexin V+ cells in the entire Ly5.1+ LSK cell population was calculated. The data from four independent experiments are shown as the means ? SD (n = 6 for each condition). **p [lt] .01.
stem1577-sup-0008-suppfig8.jpg925KFigure S8. Representative FACS profiles of the low chimerism of donor-derived hematopoietic cells engrafted in the secondary recipient mice. The BM MNCs (2.5 × 106) from the Ly5.1+ donor mice were transplanted into the primary recipient mice. The mice were sacrificed 15 weeks later, and their donor-derived Ly5.1+ BM MNCs (2 × 104) were transplanted into secondary Ly5.2+ recipient mice, with competitor BM MNCs (5 × 105) from the Ly5.2+ background mouse. Twelve weeks later, the BM cells in the secondary Ly5.2+ recipient mice were analyzed by FACS. The low chimerism (1.07%) of the Ly5.1+ populations detectable in the secondary recipient mice demonstrated multilineage differentiation, as evidenced by the presence of both Mac-1+/Gr-1+ cells and B220+ cells. The numbers in quadrants indicate the percentage of the population. The experiments were repeated twice, and the representative results of one experiment are shown.
stem1577-sup-0009-suppfig9.jpg1381KFigure S9. Schema of a summary of this study. (A): Irradiation augments the endogenous tPA level, which promotes HSC proliferation via the potentiation of the MMP-mediated release of hematopoietic factors, such as c-KitL, from BM stromal cells. However, the PAI-1 level is simultaneously upregulated in the BM compartment. PAI-1 inactivates tPA, thereby negatively regulating hematopoietic regeneration. The administration of recombinant tPA (rtPA) augments the tPA activity in the BM compartment and simultaneously induces the PAI-1 expression via a negative feedback mechanism. Therefore, hematopoietic regeneration is dependent on the balance between the levels of tPA and PAI-1. (B): Inhibition of the PAI-1 activity, either genetically or by a low-molecular-weight compound, potentiates the tPA-mediated fibrinolytic pathway, thereby promoting rapid and sustainable hematopoietic regeneration. Ob: osteoblast. EC: endothelial cell.

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