Figure 4. Cxcr41013-bearing HSCs exhibit changes in the quiescence/cycling balance in the BM. (A, left) Representative flow cytometric analyses comparing WT, +/1013, and 1013/1013 LSK frequencies in the BM. (right) Absolute numbers of LSK, LSK SLAM, LT-HSCs (LSK Flt3−CD34−CD48−CD150+), and ST-HSCs (LSK Flt3−CD34+CD48−CD150+). Data are from at least four independent experiments with 13–17 mice per group. (B) Ki-67 and DAPI costaining was used to analyze by flow cytometry the cell cycle status of LSK SLAM, LT-HSC, and ST-HSC subsets in the BM. (left) Representative flow cytometric analysis of ST-HSCs in the BM of WT, +/1013, and 1013/1013 mice. (right) Bar graphs showing the percentage of cells (DAPIlowKi-67−) in the quiescent G0 phase. Data are from at least three independent experiments with five to nine mice per group. (C) Absolute numbers of BM ST-HSCs 3.5 d after a single dose of PBS or 5-FU. Data are from three independent experiments with six mice per group. (D) Percentage of BrdU+ in ST-HSCs after a 12-d pulse period (pulse) and after 3 wk of chase (chase). Data are from two independent experiments with four to eight mice per group at each time point. (E) Expression levels of D-cyclins (Ccnd1, Ccnd2, and Ccnd3) were determined by quantitative real-time PCR in ST-HSCs sorted from the BM of WT and mutant mice. Each individual sample was run in triplicate with eight mice per group and has been standardized for β-actin expression levels. Results are expressed as percentage of decrease in mutant ST-HSCs compared with the levels obtained in WT ones (set at 100%). (F, left) Representative histograms for intracellular detection of cyclin D3 by flow cytometry in gated BM ST-HSCs from WT and mutant mice. (right) Bar graphs denote the percentage of decrease in cyclin D3 MFI values in mutant ST-HSCs compared with the levels obtained in WT ones (set at 100%). Data are from two independent experiments with six mice per group. All displayed results are represented as means ± SEM. Kruskal–Wallis H test–associated p-values (#) are indicated. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 compared with WT cells; §, P < 0.05 compared with +/1013 cells (as determined using the two-tailed Student’s t test).
Figure 4.

Cxcr41013-bearing HSCs exhibit changes in the quiescence/cycling balance in the BM. (A, left) Representative flow cytometric analyses comparing WT, +/1013, and 1013/1013 LSK frequencies in the BM. (right) Absolute numbers of LSK, LSK SLAM, LT-HSCs (LSK Flt3−CD34−CD48−CD150+), and ST-HSCs (LSK Flt3−CD34+CD48−CD150+). Data are from at least four independent experiments with 13–17 mice per group. (B) Ki-67 and DAPI costaining was used to analyze by flow cytometry the cell cycle status of LSK SLAM, LT-HSC, and ST-HSC subsets in the BM. (left) Representative flow cytometric analysis of ST-HSCs in the BM of WT, +/1013, and 1013/1013 mice. (right) Bar graphs showing the percentage of cells (DAPIlowKi-67−) in the quiescent G0 phase. Data are from at least three independent experiments with five to nine mice per group. (C) Absolute numbers of BM ST-HSCs 3.5 d after a single dose of PBS or 5-FU. Data are from three independent experiments with six mice per group. (D) Percentage of BrdU+ in ST-HSCs after a 12-d pulse period (pulse) and after 3 wk of chase (chase). Data are from two independent experiments with four to eight mice per group at each time point. (E) Expression levels of D-cyclins (Ccnd1, Ccnd2, and Ccnd3) were determined by quantitative real-time PCR in ST-HSCs sorted from the BM of WT and mutant mice. Each individual sample was run in triplicate with eight mice per group and has been standardized for β-actin expression levels. Results are expressed as percentage of decrease in mutant ST-HSCs compared with the levels obtained in WT ones (set at 100%). (F, left) Representative histograms for intracellular detection of cyclin D3 by flow cytometry in gated BM ST-HSCs from WT and mutant mice. (right) Bar graphs denote the percentage of decrease in cyclin D3 MFI values in mutant ST-HSCs compared with the levels obtained in WT ones (set at 100%). Data are from two independent experiments with six mice per group. All displayed results are represented as means ± SEM. Kruskal–Wallis H test–associated p-values (#) are indicated. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 compared with WT cells; §, P < 0.05 compared with +/1013 cells (as determined using the two-tailed Student’s t test).

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