Loss of MC5R compromises proliferation and reconstitution ability of HSCs. (A-C) Flow cytometric analysis of the cell cycle distribution of (B) LSKs and (C) LT-HSCs in the BM of WT and MC5R–/– mice 15 days after 5.0 Gy TBI (n = 6). Representative flow cytometric plots are shown (A). (D) Flow cytometric analysis of the percentage of BrdU+ cells in LSKs and LT-HSCs from the BM of WT and MC5R–/– mice 15 days after 5.0 Gy TBI (n = 6). Representative flow cytometric plots (left). (E-G) A total of 5 × 103 LSKs obtained from the BM of WT and MC5R–/– mice (CD45.2) were mixed with 5 × 105 CD45.1 BM cells and transplanted into lethally irradiated CD45.1 recipient mice. (E) The scheme of competitive BMT. (F) Flow cytometric analysis of the percentage of donor-derived cells in the PB of recipient mice 16 weeks after transplantation (n = 6). Representative flow cytometric plots (left). (G) Chimerism levels of BM, LSKs, and LT-HSCs in recipient mice at 16 weeks after transplantation (n = 6). (H) LSKs sorted from the BM of WT and MC5R–/– mice were cultured in the presence of different concentrations of α-MSH (0, 5, 50, or 500 nM). The total cell counts in the medium were counted 7 days after culture (n = 6). (I,J) A total of 1 × 106 CD45.1 BM cells were transplanted into lethally irradiated WT or MC5R–/– recipient mice (CD45.2). (I) The scheme of reciprocal BMT. (J) Flow cytometric analysis of the percentage of donor-derived cells in the PB of recipient mice at 16 weeks after transplantation (n = 6). (K,L) A total of 2 × 105 LSKs obtained from the BM of WT and MC5R–/– mice were labeled with CFSE and transplanted into lethally irradiated WT recipient mice. The percentage of CFSE+ cells in the BM of recipient mice 16 hours after transplantation was detected using flow cytometry. (K) The scheme of homing assay. (L) The homing efficiency of WT and MC5R–/– LSKs (n = 6). (B-D,F-H, J,L) unpaired t test (two-tailed). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. CFSE, 5(6)-carboxyfluorescein diacetate succinimidyl ester.
