Figure 1.
Figure 1. Survival of K3−/− chimeras and distribution of K3−/− HSPCs. (A) Kaplan-Meier survival curve of first generation K3+/+ and K3−/− FL chimeras. ***, P < 0.0001 by log-rank test. n = 41–47 per genotype; 15 independent experiments. (B) Representative FACS plots showing FL MNCs gated for lin− cells (left), expression of AA4.1 and Mac-1 on lin− cells (middle), and c-kit and Sca-1 expression on lin−AA4.1+Mac-1med cells (right). Shown are the percentages of events within the gate ± SD. n = 8–9 per genotype. (C) Total number of FL MNCs from E14.5 embryos. n = 22–23 per genotype; four independent experiments. (D) Quantification of overall frequencies (percentage of live leukocytes) of LSK cells in E14.5 FLs. Error bars represent mean percentage ± SD. n = 8–9 per genotype. (C and D) ***, P < 0.0001 by unpaired t test. (E) Total number of LSK cells in E14.5 FLs. ns, P = 0.3527 by unpaired t test. n = 8–9 per genotype. (F) Frequency of CFU-Cs in E14.5 FLs. Error bars represent mean frequency ± SD. **, P = 0.0021 by Mann-Whitney test. n = 10 per genotype; three independent experiments. (G) LTC-IC assay performed with FL MNCs by limiting dilutions. Percentage of wells without CFUs is plotted against the MNC number. The frequency of LTC-ICs is shown. P < 0.0001 by Pearson’s χ2 test. n = 4 per genotype; two independent experiments. (H) Total number of CFU-Cs in E14.5 FLs. ***, P = 0.0002 by unpaired t test. (I) Total number of LTC-ICs per FL of E14.5 embryos. ns, P > 0.05 by Mann-Whitney test. (H and I) n = 22–23 per genotype; four independent experiments. (J) Total number of MNCs from BM and whole Spl. Data are mean cell counts ± SD. ns, P > 0.05; ***, P < 0.0001 by Kruskal-Wallis test and Dunn’s multiple comparison post-test. n = 10–15 per group; 12 independent experiments. (K) Total number of B cells (B220+), T cells (CD3e+), erythroid cells (Ter119+), neutrophils (Gr-1hiMac-1+), and monocytes/macrophages (Gr-1intMac-1+) in the BM. Data are presented as box and whisker plots. The horizontal lines inside the boxes represent the median, the box edges show the lower and upper quartiles, and the whiskers indicate the minimum and maximum. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Kruskal-Wallis test and Dunn’s multiple comparison post-test. n = 10–15 per genotype; 12 independent experiments. (C, E, H, and I) Error bars represent mean cell counts ± SD. ns, not significant.

Survival of K3−/− chimeras and distribution of K3−/− HSPCs. (A) Kaplan-Meier survival curve of first generation K3+/+ and K3−/− FL chimeras. ***, P < 0.0001 by log-rank test. n = 41–47 per genotype; 15 independent experiments. (B) Representative FACS plots showing FL MNCs gated for lin− cells (left), expression of AA4.1 and Mac-1 on lin− cells (middle), and c-kit and Sca-1 expression on lin−AA4.1+Mac-1med cells (right). Shown are the percentages of events within the gate ± SD. n = 8–9 per genotype. (C) Total number of FL MNCs from E14.5 embryos. n = 22–23 per genotype; four independent experiments. (D) Quantification of overall frequencies (percentage of live leukocytes) of LSK cells in E14.5 FLs. Error bars represent mean percentage ± SD. n = 8–9 per genotype. (C and D) ***, P < 0.0001 by unpaired t test. (E) Total number of LSK cells in E14.5 FLs. ns, P = 0.3527 by unpaired t test. n = 8–9 per genotype. (F) Frequency of CFU-Cs in E14.5 FLs. Error bars represent mean frequency ± SD. **, P = 0.0021 by Mann-Whitney test. n = 10 per genotype; three independent experiments. (G) LTC-IC assay performed with FL MNCs by limiting dilutions. Percentage of wells without CFUs is plotted against the MNC number. The frequency of LTC-ICs is shown. P < 0.0001 by Pearson’s χ2 test. n = 4 per genotype; two independent experiments. (H) Total number of CFU-Cs in E14.5 FLs. ***, P = 0.0002 by unpaired t test. (I) Total number of LTC-ICs per FL of E14.5 embryos. ns, P > 0.05 by Mann-Whitney test. (H and I) n = 22–23 per genotype; four independent experiments. (J) Total number of MNCs from BM and whole Spl. Data are mean cell counts ± SD. ns, P > 0.05; ***, P < 0.0001 by Kruskal-Wallis test and Dunn’s multiple comparison post-test. n = 10–15 per group; 12 independent experiments. (K) Total number of B cells (B220+), T cells (CD3e+), erythroid cells (Ter119+), neutrophils (Gr-1hiMac-1+), and monocytes/macrophages (Gr-1intMac-1+) in the BM. Data are presented as box and whisker plots. The horizontal lines inside the boxes represent the median, the box edges show the lower and upper quartiles, and the whiskers indicate the minimum and maximum. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Kruskal-Wallis test and Dunn’s multiple comparison post-test. n = 10–15 per genotype; 12 independent experiments. (C, E, H, and I) Error bars represent mean cell counts ± SD. ns, not significant.

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