Figure 3.
Effects of elevated RSPO3 levels on HSCs and early B-progenitors. (A) Representative fluorescence-activated cell sorting (FACS) plot and corresponding bar graphs quantifying the relative abundance of megakaryocyte-erythrocyte progenitors (MEP), granulocyte-macrophage progenitors (GMP), and common myeloid progenitors (CMP) among Lin–Sca-1–Il-7R–c-kit+ bone marrow cells from n = 7 control and n = 10 RSPO3 animals. Bar graphs depicting relative abundance of megakaryocytes (CD41+CD45+FSChigh) (B) and myeloid progenitors (CD11b+Gr-1+) (C) among CD45+ bone marrow cells from n = 3 control and n = 3 to 4 RSPO3 animals. (D) Representative FACS plots and corresponding bar graphs quantifying the relative abundance of LSK (Lin–c-kit+Sca-1+) cells in the bone marrow of control and RSPO3 mice; n = 7 per group. (E) Bar graphs depicting the proportion of LSK cells from control and RSPO3 animals at various stages of cell cycle determined by using 5-bromodeoxyuridine incorporation; n = 5 per group. (F) Bar graphs showing the proportion of long-term HSCs (LT-HSC, CD150+CD48–) and restricted hematopoietic progenitor cell fractions (HPC-1, CD150–CD48+; HPC-2, CD150+CD48+) among LSK cells from n = 6 control and n = 9 RSPO3 animals. (G) Bar graphs depicting the relative abundance of 3 major lymphocyte subpopulations in peripheral blood of n = 7 control and n = 3 RSPO3 mice. (H) Representative FACS plots and corresponding bar graph showing the relative abundance of pro–/pre–B cells (CD45RA+IgM–), immature B cells (CD45RA+IgM+), and mature B cells (CD45RA++IgM+) among mononuclear cells from n = 7 control and n = 6 RSPO3 animals. (I) Bar graph depicting the absolute number of pro–/pre–B cells (CD45RA+IgM–) per microliter of peripheral blood from control and RSPO3 mice; n = 3 per group. (J) Bar graph showing the number of colony-forming units (CFU) (pre–B cell) after culturing equal numbers of total bone marrow cells from n = 10 control and n = 4 RSPO3 mice for 10 days in methylcellulose. (K) Representative FACS plots and corresponding bar graph quantifying the combined relative number of pro–B cells (CD43+CD19–) and pre–B cells (CD43–CD19+) among CD45RA+IgM– B-progenitors from control and RSPO3 mice; n = 10 per group. (L) Bar graph depicting the absolute number of pro–B cells (CD45RA+IgM–CD43+CD19–) per microliter of peripheral blood from control and RSPO3 mice; n = 3 per group. (M) Bar graph depicting the percent common lymphoid progenitors (CLP; Lin–Il-7R+) among viable bone marrow cells from control and RSPO3 animals; n = 12 per group. Bone marrow was analyzed 1 month after tamoxifen induction, and data are represented as mean ± standard error of the mean. Two-tailed unpaired t test. APC, allophycocyanin; BV, brilliant violet; Ct, control; FcγRII/III, Fc gamma receptor II/III; FITC, fluorescein isothiocyanate; IgM, immunoglobulin M; n.s., not significant (P > .05); PE, phycoerythrin; RSPO3, tamoxifen-induced animals.

Effects of elevated RSPO3 levels on HSCs and early B-progenitors. (A) Representative fluorescence-activated cell sorting (FACS) plot and corresponding bar graphs quantifying the relative abundance of megakaryocyte-erythrocyte progenitors (MEP), granulocyte-macrophage progenitors (GMP), and common myeloid progenitors (CMP) among Lin–Sca-1–Il-7R–c-kit+ bone marrow cells from n = 7 control and n = 10 RSPO3 animals. Bar graphs depicting relative abundance of megakaryocytes (CD41+CD45+FSChigh) (B) and myeloid progenitors (CD11b+Gr-1+) (C) among CD45+ bone marrow cells from n = 3 control and n = 3 to 4 RSPO3 animals. (D) Representative FACS plots and corresponding bar graphs quantifying the relative abundance of LSK (Lin–c-kit+Sca-1+) cells in the bone marrow of control and RSPO3 mice; n = 7 per group. (E) Bar graphs depicting the proportion of LSK cells from control and RSPO3 animals at various stages of cell cycle determined by using 5-bromodeoxyuridine incorporation; n = 5 per group. (F) Bar graphs showing the proportion of long-term HSCs (LT-HSC, CD150+CD48–) and restricted hematopoietic progenitor cell fractions (HPC-1, CD150–CD48+; HPC-2, CD150+CD48+) among LSK cells from n = 6 control and n = 9 RSPO3 animals. (G) Bar graphs depicting the relative abundance of 3 major lymphocyte subpopulations in peripheral blood of n = 7 control and n = 3 RSPO3 mice. (H) Representative FACS plots and corresponding bar graph showing the relative abundance of pro–/pre–B cells (CD45RA+IgM–), immature B cells (CD45RA+IgM+), and mature B cells (CD45RA++IgM+) among mononuclear cells from n = 7 control and n = 6 RSPO3 animals. (I) Bar graph depicting the absolute number of pro–/pre–B cells (CD45RA+IgM–) per microliter of peripheral blood from control and RSPO3 mice; n = 3 per group. (J) Bar graph showing the number of colony-forming units (CFU) (pre–B cell) after culturing equal numbers of total bone marrow cells from n = 10 control and n = 4 RSPO3 mice for 10 days in methylcellulose. (K) Representative FACS plots and corresponding bar graph quantifying the combined relative number of pro–B cells (CD43+CD19–) and pre–B cells (CD43–CD19+) among CD45RA+IgM– B-progenitors from control and RSPO3 mice; n = 10 per group. (L) Bar graph depicting the absolute number of pro–B cells (CD45RA+IgM–CD43+CD19–) per microliter of peripheral blood from control and RSPO3 mice; n = 3 per group. (M) Bar graph depicting the percent common lymphoid progenitors (CLP; Lin–Il-7R+) among viable bone marrow cells from control and RSPO3 animals; n = 12 per group. Bone marrow was analyzed 1 month after tamoxifen induction, and data are represented as mean ± standard error of the mean. Two-tailed unpaired t test. APC, allophycocyanin; BV, brilliant violet; Ct, control; FcγRII/III, Fc gamma receptor II/III; FITC, fluorescein isothiocyanate; IgM, immunoglobulin M; n.s., not significant (P > .05); PE, phycoerythrin; RSPO3, tamoxifen-induced animals.

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