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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Nat Med. 2015 Dec 14;22(1):78–83. doi: 10.1038/nm.4010

Figure 3.

Figure 3

SC clearance by treatment with ABT263 or GCV rejuvenates HSCs after TBI. (a) Experimental design for b–f. Sham-irradiated (Ctl) and TBI-treated young male C57BL/6 mice were administered vehicle (Veh) or ABT263 (ABT) and analyzed as indicated. (b,c) Quantification of changes in Cdkn2a mRNA levels (b, left; n = 3 biological replicates) and the percentage of SA–β-gal+ cells (b, right; n = 4 mice per group) in sorted HSCs, and the number of day 35 cobblestone area–forming cells (CAFCs) in bone marrow cells (BMCs; c; n = 4 mice per group). #P < 0.05 versus Ctl mice; &P < 0.05 versus vehicle-treated TBI mice; two-way ANOVA; n.d., not detectable. (d) Scheme for competitive and serial bone marrow transplantation (BMT; see Online Methods for details). (e,f) Representative flow cytometry plots (e) and quantification (f) of donor-derived total white blood cells (CD45.2+; e, top; f, top left), and T cells (T; CD45.2+Thy-1.2+), B cells (B; CD45.2+B220+) and myeloid cells (M cells; CD45.2+CD11b/Gr-1+) (e, bottom; f) in the peripheral blood (PB) of recipient mice after primary BMT (n = 6 recipients per group). Donor-cell engraftment in the PB of secondary recipients is shown in Supplementary Figure 6. (g) Experimental design for h–j. Sham-irradiated (Ctl) and TBI-exposed young male p16–3MR mice were treated with vehicle or GCV and analyzed as indicated. (h) Quantification of the percentages of SA–β-gal+ cells in sorted HSCs from mice treated as outlined in g (Ctl mice: vehicle-treated, n = 7; GCV-treated, n = 7; TBI mice: vehicle-treated, n = 11; GCV-treated, n = 12). (i) Quantification of the number of day 35 CAFCs in BMCs (Ctl mice: vehicle-treated, n = 3; GCV-treated, n = 3; TBI mice: vehicle-treated, n = 7; GCV-treated, n = 6). (j) Quantification of the percentages of donor-derived total cells, T cells, B cells and M cells in the PB of primary recipients (Ctl donors: vehicle-treated, n = 3 recipients; GCV-treated, n = 5 recipients; TBI donors: vehicle-treated, n = 7 recipients; GCV-treated, n = 9 recipients). Throughout, data are means ± s.e.m. In f,j, #P < 0.05 versus recipients of donor-derived cells from Ctl mice; &P < 0.05 versus recipients of donor-derived cells from vehicle-treated TBI mice; in h,i, #P < 0.05 versus Ctl mice; &P < 0.05 versus vehicle-treated TBI mice; two-way ANOVA; n.d., not detectable.