![Figure 6. Kindlin-3 retains active HSCs and HPCs in the BM. (A) Design showing the generation of mix chimeras. E14.5 FL cells (CD45.2+) were mixed with WT congenic B6.SJL E14.5 FL cells (CD45.1+) and transplanted to lethally irradiated (C57BL/6 × congenic B6.SJL) F1 generation recipient mice (CD45.1+/CD45.2+). (B) Representative FACS plots of PB from mix chimeras 4 mo after transplantation. The top panels show whole leukocytes, and the bottom panels show lin− cells from CD45.2+ and CD45.1+ populations gated for c-kit against Sca-1. Numbers represent mean overall frequencies (percentage of live PB leukocytes) ± SD. n = 6–8 per genotype; six independent experiments. (C) G-CSF–induced mobilization of LSK cells in mix chimeras. The histogram shows the mean number of LSK cells per milliliter PB ± SD. ns, P > 0.05; **, P < 0.01; and ***, P < 0.001 by a one-way analysis of variance followed by Tukey’s multiple comparison test. n = 5–6 per genotype and treatment. (D–F) Percentage of donor-derived LSK CD150+CD48−CD34− (D), LSK CD150+CD48− (E), and LSK CD150−CD48+ populations (F) in the BM of mix chimeras at the indicated times after transplantation. Dots show the mean percentage ± SD. ns, P > 0.05; ***, P < 0.001 by unpaired t test. n = 4–11 per genotype and time point; two independent experiments. (G) LRC assay with mix chimeras. Percentages of BrdU LRCs in the indicated populations after 45–50 d of chase phase. Total LSK (*, P = 0.0286), LSK CD150−CD48+ (*, P = 0.0286), LSK CD150+CD48− (ns, P = 0.0571), and LSK CD150+CD34− cells (ns, P = 0.1143) by Mann-Whitney test. Error bars represent mean percentage ± SD. n = 4 per genotype; two independent experiments. (H) Percentage of Ki67+ donor cells (CD45.2+) in the indicated populations. ns, P > 0.05 by unpaired t test. n = 8 per genotype; three independent experiments. (I–K) Percentage of donor-derived LSK CD150+CD48−CD34− (I), LSK CD150+CD48− (J), and LSK CD150−CD48+ (K) populations in BM from K3+/+Rosa26Cre-ERT2 (blue) and K3fl/flRosa26Cre-ERT2 (red) mix chimeras untreated (Ctrl.), treated with tamoxifen (TAM PBS), or treated with tamoxifen and 5-FU (TAM 5-FU). The histogram shows the mean percentage ± SD. ns, P > 0.05; *, P < 0.05; and ***, P < 0.0006 by unpaired t test. n = 4–6 per genotype and treatment. (L) Competitive repopulation assay with BM LSK CD150+CD45.2+ cells from mix chimeras. Recipients received LSK CD150+CD45.2+ cells and host-type whole Spl cells. 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 individual recipients at the indicated times after transplantation. Significant differences in mean lineage engraftment are indicated (4 wk after transplantation: leukocytes [ns, P = 0.8707] by unpaired t test; myeloid cells [**, P = 0.0093], B cells [*, P = 0.0337], and T cells [**, P = 0.0074] by Mann-Whitney test; 8 and 12 wk after transplantation: leukocytes: ***, P < 0.0001 by unpaired t test; myeloid, B, and T cells: ***, P < 0.0001 [or P = 0.0006 for myeloid cells after 8 wk] by Mann-Whitney test). M) Mean percentage ± SD of donor-derived HSPCs in BM of second generation recipients 12 wk after transplantation. ***, P < 0.0001 by Mann-Whitney test. (L and M) n = 14–17; four independent experiments. ns, not significant; Wt, wild type.](https://cdn.rupress.org/rup/content_public/journal/jem/212/9/10.1084_jem.20150269/6/jem_20150269_fig6.jpeg?Expires=1771570590&Signature=1wC6OedhbfeorWQ4zoydCRLMLxWnZgZijueb1U9jrsr28TAQyHqpgFjelv9i6HFQ6hGTt7-yYkPp9oVKvUt8Y5lsOAoCJN~BQDLB1ns5qqB5tPDVHsXW9XYCMHvIDKNawaSOFRGf~gfkcCDw19Zm5WHGCQhTgoRwRE2F3u~507fDBTQlQdjBM4msRyxivzqyWOAmSqmr4-wRNIEgkBR2QSjfpWvjSedWbixrpzRTNf47oNgvzR-w5b4KddNFHwGwz8UulxMq~bE~PR6TUF8kHxjPqP4cKG-92Xjgeckr2LuI754lvlcCBFs5ZCf8ro7xUfAXP-z3rzfuSqXqlaNcqA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Kindlin-3 retains active HSCs and HPCs in the BM. (A) Design showing the generation of mix chimeras. E14.5 FL cells (CD45.2+) were mixed with WT congenic B6.SJL E14.5 FL cells (CD45.1+) and transplanted to lethally irradiated (C57BL/6 × congenic B6.SJL) F1 generation recipient mice (CD45.1+/CD45.2+). (B) Representative FACS plots of PB from mix chimeras 4 mo after transplantation. The top panels show whole leukocytes, and the bottom panels show lin− cells from CD45.2+ and CD45.1+ populations gated for c-kit against Sca-1. Numbers represent mean overall frequencies (percentage of live PB leukocytes) ± SD. n = 6–8 per genotype; six independent experiments. (C) G-CSF–induced mobilization of LSK cells in mix chimeras. The histogram shows the mean number of LSK cells per milliliter PB ± SD. ns, P > 0.05; **, P < 0.01; and ***, P < 0.001 by a one-way analysis of variance followed by Tukey’s multiple comparison test. n = 5–6 per genotype and treatment. (D–F) Percentage of donor-derived LSK CD150+CD48−CD34− (D), LSK CD150+CD48− (E), and LSK CD150−CD48+ populations (F) in the BM of mix chimeras at the indicated times after transplantation. Dots show the mean percentage ± SD. ns, P > 0.05; ***, P < 0.001 by unpaired t test. n = 4–11 per genotype and time point; two independent experiments. (G) LRC assay with mix chimeras. Percentages of BrdU LRCs in the indicated populations after 45–50 d of chase phase. Total LSK (*, P = 0.0286), LSK CD150−CD48+ (*, P = 0.0286), LSK CD150+CD48− (ns, P = 0.0571), and LSK CD150+CD34− cells (ns, P = 0.1143) by Mann-Whitney test. Error bars represent mean percentage ± SD. n = 4 per genotype; two independent experiments. (H) Percentage of Ki67+ donor cells (CD45.2+) in the indicated populations. ns, P > 0.05 by unpaired t test. n = 8 per genotype; three independent experiments. (I–K) Percentage of donor-derived LSK CD150+CD48−CD34− (I), LSK CD150+CD48− (J), and LSK CD150−CD48+ (K) populations in BM from K3+/+Rosa26Cre-ERT2 (blue) and K3fl/flRosa26Cre-ERT2 (red) mix chimeras untreated (Ctrl.), treated with tamoxifen (TAM PBS), or treated with tamoxifen and 5-FU (TAM 5-FU). The histogram shows the mean percentage ± SD. ns, P > 0.05; *, P < 0.05; and ***, P < 0.0006 by unpaired t test. n = 4–6 per genotype and treatment. (L) Competitive repopulation assay with BM LSK CD150+CD45.2+ cells from mix chimeras. Recipients received LSK CD150+CD45.2+ cells and host-type whole Spl cells. 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 individual recipients at the indicated times after transplantation. Significant differences in mean lineage engraftment are indicated (4 wk after transplantation: leukocytes [ns, P = 0.8707] by unpaired t test; myeloid cells [**, P = 0.0093], B cells [*, P = 0.0337], and T cells [**, P = 0.0074] by Mann-Whitney test; 8 and 12 wk after transplantation: leukocytes: ***, P < 0.0001 by unpaired t test; myeloid, B, and T cells: ***, P < 0.0001 [or P = 0.0006 for myeloid cells after 8 wk] by Mann-Whitney test). M) Mean percentage ± SD of donor-derived HSPCs in BM of second generation recipients 12 wk after transplantation. ***, P < 0.0001 by Mann-Whitney test. (L and M) n = 14–17; four independent experiments. ns, not significant; Wt, wild type.
