Figure 4.
Figure 4. K3−/− HSCs remain hyperactive and accumulate in the PB. (A and B) PolyI:C and 5-FU double treatment of K3+/+ and K3−/− chimeras. (A) Diagram showing drug combinations and timing. (B) Kaplan-Meier survival curves. ***, P < 0.0001 by log-rank test. n = 16–22 per treatment group; five independent experiments. (C) In vivo BrdU uptake assay shown as mean percentage ± SD of BrdU+ cells. BrdU levels were measured 12–14 h after injection in LS−K+ (LK; P = 0.4078 by unpaired t test), LSK (***, P = 0.0009 by unpaired t test), LSK CD150+CD48− (**, P = 0.0014 by Mann-Whitney test), and LSK CD150+CD34− cells (***, P = 0.0004 by Mann-Whitney test). n = 6–14 per genotype; six independent experiments. (D) Percentage of Ki67+ BM LSK cells indicated as mean values ± SD. **, P = 0.0022 by paired t test. n = 18 per genotype; 12 independent experiments. (E) Relative expression of p21, p27, and p57 mRNA in sorted CD150+ LSK cells analyzed by quantitative PCR (mean ± SD). ***, P = 0.0007 by unpaired t test. n = 6–7 per genotype; six independent experiments. (F) Mean percentage ± SD of BrdU LRCs in the indicated populations after 45 d of chase phase: total LSK cells (***, P < 0.0001 by unpaired t test), LSK CD150−CD48+ (***, P < 0.0001), LSK CD150+CD48+ (***, P = 0.0006), LSK CD150+CD48− (**, P = 0.0014), and LSK CD150+CD34− (*, P = 0.0350) by Mann-Whitney test. n = 7–8 per genotype; three independent experiments. (G) Representative FACS plots showing the expression of c-kit and Sca-1 on lin− cells from Spl and PB. The frequencies of LS−K+ (orange boxes) and LSK cells (green boxes) are shown (percentages of live leukocytes ± SD). n = 24–26 per genotype; 10 independent experiments. (H) Quantification of LS−K+ (LK) and LSK cells in Spl. (ns, P > 0.05; **, P = 0.0031) and PB (***, P < 0.0001). Statistics by Mann-Whitney test. Error bars represent mean cell counts ± SD. n = 25–26 per genotype; 10 independent experiments. (I) Frequency of CFU-Cs shown as mean cell counts ± SD in CD45.2+ cells from Spl (*, P = 0.0286) and PB (*, P = 0.0294). Statistics by Mann-Whitney test. n = 4 per genotype; three independent experiments. (J) Multilineage engraftment potential of PB cells isolated from chimeras. Shown are percentages of donor-derived whole leukocytes (CD45+), myeloid cells (Gr-1+, Mac-1+, and Gr-1+Mac-1+), B cells (B220+), and T cells (CD3e+) from PB at the indicated times after transplantation. Data are plotted for individual recipients. n = 5 for K3+/+ (blue lines) and n = 19 for K3−/− (red lines); three independent experiments. Differences in mean lineage engraftment 16 wk after transplantation are indicated (leukocytes: *, P = 0.0157; myeloid cells: **, P = 0.0085; B cells: **, P = 0.0028; and T cells: ***, P = 0.0008). Statistics by Mann-Whitney test. (K) Mobilization of LSK cells in chimeras shown as LSK cells per milliliter of blood after G-CSF or PBS treatment. Significance at day 6 is indicated (K3+/+ vs. K3−/− with G-CSF: ***, P < 0.0001; K3+/+ vs. K3−/− with PBS: **, P = 0.0049 by unpaired t test). n = 3–5 per genotype; four independent experiments. ns, not significant.

K3−/− HSCs remain hyperactive and accumulate in the PB. (A and B) PolyI:C and 5-FU double treatment of K3+/+ and K3−/− chimeras. (A) Diagram showing drug combinations and timing. (B) Kaplan-Meier survival curves. ***, P < 0.0001 by log-rank test. n = 16–22 per treatment group; five independent experiments. (C) In vivo BrdU uptake assay shown as mean percentage ± SD of BrdU+ cells. BrdU levels were measured 12–14 h after injection in LS−K+ (LK; P = 0.4078 by unpaired t test), LSK (***, P = 0.0009 by unpaired t test), LSK CD150+CD48− (**, P = 0.0014 by Mann-Whitney test), and LSK CD150+CD34− cells (***, P = 0.0004 by Mann-Whitney test). n = 6–14 per genotype; six independent experiments. (D) Percentage of Ki67+ BM LSK cells indicated as mean values ± SD. **, P = 0.0022 by paired t test. n = 18 per genotype; 12 independent experiments. (E) Relative expression of p21, p27, and p57 mRNA in sorted CD150+ LSK cells analyzed by quantitative PCR (mean ± SD). ***, P = 0.0007 by unpaired t test. n = 6–7 per genotype; six independent experiments. (F) Mean percentage ± SD of BrdU LRCs in the indicated populations after 45 d of chase phase: total LSK cells (***, P < 0.0001 by unpaired t test), LSK CD150−CD48+ (***, P < 0.0001), LSK CD150+CD48+ (***, P = 0.0006), LSK CD150+CD48− (**, P = 0.0014), and LSK CD150+CD34− (*, P = 0.0350) by Mann-Whitney test. n = 7–8 per genotype; three independent experiments. (G) Representative FACS plots showing the expression of c-kit and Sca-1 on lin− cells from Spl and PB. The frequencies of LS−K+ (orange boxes) and LSK cells (green boxes) are shown (percentages of live leukocytes ± SD). n = 24–26 per genotype; 10 independent experiments. (H) Quantification of LS−K+ (LK) and LSK cells in Spl. (ns, P > 0.05; **, P = 0.0031) and PB (***, P < 0.0001). Statistics by Mann-Whitney test. Error bars represent mean cell counts ± SD. n = 25–26 per genotype; 10 independent experiments. (I) Frequency of CFU-Cs shown as mean cell counts ± SD in CD45.2+ cells from Spl (*, P = 0.0286) and PB (*, P = 0.0294). Statistics by Mann-Whitney test. n = 4 per genotype; three independent experiments. (J) Multilineage engraftment potential of PB cells isolated from chimeras. Shown are percentages of donor-derived whole leukocytes (CD45+), myeloid cells (Gr-1+, Mac-1+, and Gr-1+Mac-1+), B cells (B220+), and T cells (CD3e+) from PB at the indicated times after transplantation. Data are plotted for individual recipients. n = 5 for K3+/+ (blue lines) and n = 19 for K3−/− (red lines); three independent experiments. Differences in mean lineage engraftment 16 wk after transplantation are indicated (leukocytes: *, P = 0.0157; myeloid cells: **, P = 0.0085; B cells: **, P = 0.0028; and T cells: ***, P = 0.0008). Statistics by Mann-Whitney test. (K) Mobilization of LSK cells in chimeras shown as LSK cells per milliliter of blood after G-CSF or PBS treatment. Significance at day 6 is indicated (K3+/+ vs. K3−/− with G-CSF: ***, P < 0.0001; K3+/+ vs. K3−/− with PBS: **, P = 0.0049 by unpaired t test). n = 3–5 per genotype; four independent experiments. ns, not significant.

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