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. 2015 Apr 6;212(4):525–538. doi: 10.1084/jem.20141438

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© 2015 Tesio et al.

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Figure 1. — CYLD catalytic activity is required to preserve HSC activity. (A) Expression of CYLD in label-retaining dormant and non–label-retaining homeostatic HSCs. (B) Cre-mediated recombination of the CYLD^932flox/flox allele generates a truncated and catalytically inactive protein. (C and D) Analysis of BM SKLCD150⁺CD48⁻CD34⁻ in CYLD^932flox/flox and CYLD^Δ932Mx mice by flow cytometry. (E) Experimental scheme to generate and analyze competitive BM chimeras (i.v. injection) normalized to equal numbers of phenotypic HSCs shown in F–I. (F) Donor-derived myeloid chimerism in the peripheral blood of recipient mice. (G–I) BM of recipient mice (18 wk after transplant) analyzed for donor-derived HSCs (G and H) and myeloid, B, and T cells (I). (J and K) Peripheral blood myeloid (J) and BM HSC chimerism (K) of mice transplanted with an equivalent ratio of test/competitor total BM cells. (L) Similar analysis as shown in E, but transplantation was performed by intrafemoral injection. Donor-derived HSC chimerism in the BM of recipients is shown 18 wk after transplant. (M) Experimental scheme to generate competitive BM chimeras in which Cre-mediated recombination was induced by four pI-pC injections after stable engraftment (16 wk) has been achieved in the chimeras. (N) Analysis of donor-derived HSCs chimerism 18 wk after pI-pC induction. Results are shown of two (F–I: 20/21; J and K: 11/12; L: 9/12; N: 13/12) or three (A: 6/6; C and D: 14/7) independent experiments, with the numbers of analyzed control/mutant mice indicated in parentheses. Error bars indicate SEM. *, P < 0.05; ***, P < 0.001.