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Blood, Vol. 111, Issue 4, 1894-1902, February 15, 2008
The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress
Blood Daria et al.
111: 1894
Supplemental materials for: Daria et al
Files in this Data Supplement:
- Figure S1. Deletion of Rb in Vav-Cre Rb KO animals (JPG, 55.2 KB)
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(A) Breeding scheme. (B)Deletion of Rb in distinct tissues, detected by PCR analysis (flox= floxed allele, delflox= deleted allele). (C) Deletion of Rb in individual CFCs, detected by PCR analysis.
- Figure S2. Altered contribution of distinct hematopoietic cell lineages in peripheralblood (PB) and spleen (JPG, 69 KB)
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(A) Blood smears from control and Vav-Cre Rb KO animals, identifying nucleated red blood cells and reticulocytes. (B) Flow cytometric analyses of PB cells from control and Vav-Cre Rb KO animals for the total number of cell with surface markers associated with myeloid cells (Mac-1, Gr-1), B-cells (B220), and T-cells (CD3e), n=5 for both control and Vav-Cre Rb KO. Shown are mean values ± 1 SEM, * = p<0.05.
- Figure S3. Reduced function of Vav-Cre Rb KO HSPCs upon transplantation (JPG, 100 KB)
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(A) Experimental setup for additional transplants into NOD-SCID animals: BM cells from 4 control and 4 Vav-Cre Rb KO animals were pooled and transplanted at 1 × 106 BM cells into irradiated (3Gy) NOD/SCID animals. Chimerism was analyzed in PB 3 and 6 weeks post transplant. (B) Animals transplanted with BM cells from Vav-Cre Rb KO animals present with a significantly reduced donor chimerism in PB (n=5, * = p<0.05). (C) Experimental setup for additional competitive transplants into B6.SJL (BoyJ) animals. BM cells from 4 individual donors (control or Vac-Cre Rb KO animals) identical to the donors in the NOD-SCID experiments presented in part B were individually competitively transplanted into 3 recipient animals each (total of 24 recipients). Chimerism in PB was analyzed 3 and 6 weeks post transplant by flow cytometry. (D) Animals transplanted with BM cells from Vav-Cre Rb KO animals present with a significantly reduced donor chimerism. * = p<0.05. (E) Equal number of LIN-cells from spleen from control and Vav-Cre Rb KO animals were transplanted into lethally irradiated C57BL/6 CD45.1 positive animals. The contribution of control and Vav-Cre Rb KO cells to PB up to 25 weeks post-transplant (chimerism in PB) was determined by flow cytometric analyses (n=4 recipients for control and n=4 recipients for Vav-Cre Rb KO). Recipient animals where sacrificed 27 weks post transplant and 2 × 106 BM cell individually transplanted into secondary recipients. The contribution of control and Vav-Cre Rb KO cells to PB was analyzed 6 weeks post transplant. Vav-Cre Rb KO cells present initially with a reduction in reconstitution potential, demonstrated by lower chimerism between 3 to 27 weeks, and were never able to fully reconstitute the recipient animal. This corresponds to a severe defect in the repopulating ability when transplanted into a fully ablated animal. The contribution by Vav-Cre Rb KO cells is further approximately 2 fold diminished upon secondary transplantation, supporting our hypothesis that upon stress/transplantation HPCs as well as HSCs devoid of Rb are impaired in their function. Shown are mean values ± 1 SEM, * = p<0.05.
- Figure S4. Relative frequency of HPCs (L-S-K+) andHSCs (L-S+K+) in spleen of control and Vav-Cre RbKO animals (JPG, 44.4 KB)
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Determined by FACS analysis as described in Material and Methods (n=5 for control and n=7 for Vav-Cre Rb KO). Please compare this data also to the total number of HPCs and HSCs in the spleen, presented in Figure 7. * = p<0.05.
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