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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 4.

Figure 4

SC clearance by ABT263 treatment rejuvenates HSCs and MuSCs in normally aged mice. (a) Representative whole-body luminescent images (left) and quantification of luminescence (right) 1 d after 21- to 22-month-old male p16-3MR mice (aged) received vehicle (Veh; n = 7) or ABT263 (ABT; n = 8), according to the scheme shown in Figure 3a. 2-month-old male p16-3MR mice (young; n = 5) without treatment and a WT C57BL/6 mouse were used as controls. The vertical color bar indicates luminescence-signal strength. #P < 0.05 versus young mice, &P < 0.05 versus vehicle-treated aged mice; unpaired Student’s t-test. Scale bars, 15 mm. (b–j) Aged (21- to 22-month-old) male C57BL/6 mice received vehicle (Veh) or ABT263 (ABT) according to the scheme shown in Figure 3a and were analyzed 1 week after the treatment; 2-month-old male C57BL/6 mice without treatment (young) were used as controls. (b) Quantification of changes in mRNA levels of Cdkn2a, Tnfa and Ccl5 in the lungs of aged (Veh, n = 11; ABT, n = 10) and young (n = 9) mice. (c) Scheme for competitive and serial BMT using sorted long-term HSCs (LT-HSCs; CD34−CD48−CD150+Lin−Sca1+c-Kit+). (d) Quantification of the percentages of donor-derived total cells, T cells, B cells and M cells in the PB of primary recipients (recipients of donor cells from: young, n = 11; vehicle-treated aged, n = 15; ABT263-treated aged, n = 13; from two independent BMT experiments). Donor cell engraftment in the PB of secondary recipients is shown in Supplementary Figure 10c. (e) Gating strategy for analysis and isolation of MuSCs by flow cytometry (top) and representative micrographs showing immunostaining for the myogenic transcription factor Pax7 (a biomarker for MuSCs) (bottom). IF, immunofluorescence; MCFC, myogenic colony-forming cell. Scale bars, 20 µm. (f) Quantification of MuSCs in muscle tissue. (g) Quantification of primary (left) and secondary (right) MCFCs in sorted MuSCs. (h,i) Quantification of p16+ cells (h) and phosphorylated p38+ (p-p38+) (i) cells in MuSCs. (j) Quantification of the average number of γ-H2AX foci in MuSCs. n = 5, 6, and 6 mice per group for Young, Aged + Veh, and Aged + ABT, respectively, for f,h,i,j; 8, 12, and 13 mice per group for g. Throughout, data are means ± s.e.m. In d, #P < 0.05 versus recipients of donor cells from young mice, &P < 0.05 versus recipients of donor cells from vehicle-treated aged mice; in g–j, #P < 0.05 versus young mice, &P < 0.05 versus vehicle-treated aged mice; unpaired Student’s t-test.