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Additional Supporting Information may be found in the online version of this article.

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sc-12-0678_sm_SupplData.pdf59KSupplementary Data
sc-12-0678_sm_SupplFigure1a.pdf553KSupplementary Figure 1. Alcam expression marks a subset of CMP cells. (A) Representative FACS plot of sorting Alcam+ CMP and Alcam- CMP subsets (top) and enrichment of PreMegE and PreGM subsets using additional markers including CD105 and CD150 (bottom). (B) Representative FACS plot (top) and morphology of cytospin preparation (bottom) for each type of colonies (Mixed, MegE and GM).
sc-12-0678_sm_SupplFigure1b.pdf429KSupplementary Figure 1. Alcam expression marks a subset of CMP cells. (A) Representative FACS plot of sorting Alcam+ CMP and Alcam- CMP subsets (top) and enrichment of PreMegE and PreGM subsets using additional markers including CD105 and CD150 (bottom). (B) Representative FACS plot (top) and morphology of cytospin preparation (bottom) for each type of colonies (Mixed, MegE and GM).
sc-12-0678_sm_SupplFigure2a.tif2024KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure2b.tif503KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure2c.tif92KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure3a.tif658KSupplementary Figure 3. Alcam- deficient cells show robust transient repopulation but reduced long- term engraftment activity. (A) Schematic of limiting- dilution transplantation experimental design. Diluting numbers (2 × 105, 4 × 104, 8 × 103) of unfractionated WT or KO cells (CD45.2+) were transplanted into lethally irradiated CD45.1+ congenic recipients (n=7 or 8) together with JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal 2 × 105 CD45.1+ radio- protective cells. Donor chimerism was assessed every 4 weeks and BM engraftment was analyzed 16 weeks after transplantation. Engraftment was considered positive if CD45.2+ donor- derived B cells (B220+ ), T cells (CD3+ ), and myeloid cells (Gr1+ and/ or CD11b+) in peripheral blood all exceeded 1% at 16 weeks post- transplantation. (B) Time course of CD45.2+ chimerism in mice transplanted with 2 × 105 donor cells (WT: n=7; KO: n=7). (C) Time course of CD45.2+ donor chimerism in mice that received 4 × 104 donor cells (WT: n=8; KO: n=8; p=0.0119). (D) Time course of CD45.2+ donor chimerism in mice that received 8 × 103 donor cells (WT: n=8; KO: n=8).
sc-12-0678_sm_SupplFigure3b.tif350KSupplementary Figure 3. Alcam- deficient cells show robust transient repopulation but reduced long- term engraftment activity. (A) Schematic of limiting- dilution transplantation experimental design. Diluting numbers (2 × 105, 4 × 104, 8 × 103) of unfractionated WT or KO cells (CD45.2+) were transplanted into lethally irradiated CD45.1+ congenic recipients (n=7 or 8) together with JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal 2 × 105 CD45.1+ radio- protective cells. Donor chimerism was assessed every 4 weeks and BM engraftment was analyzed 16 weeks after transplantation. Engraftment was considered positive if CD45.2+ donor- derived B cells (B220+ ), T cells (CD3+ ), and myeloid cells (Gr1+ and/ or CD11b+) in peripheral blood all exceeded 1% at 16 weeks post- transplantation. (B) Time course of CD45.2+ chimerism in mice transplanted with 2 × 105 donor cells (WT: n=7; KO: n=7). (C) Time course of CD45.2+ donor chimerism in mice that received 4 × 104 donor cells (WT: n=8; KO: n=8; p=0.0119). (D) Time course of CD45.2+ donor chimerism in mice that received 8 × 103 donor cells (WT: n=8; KO: n=8).
sc-12-0678_sm_SupplFigure4a.tif1468KSupplementary Figure 4. Alcam- deficient HSCs are able to home to BM and localize to endosteum. (A) Representative FACS plot for gating eFour670- labeled test cells and Violet- labeled input control cells. (B) Relative amount of eFour670- labeled Alcam−/− (KO) or WT LSK cells detected in BM, spleen (SP), peripheral blood (PB) and liver (LV) 4 hours after transplantation. Data was normalized with co- injected input control cells stained with CellTrace Violet (Invitrogen). Each dot represent an individual mouse, lines indicate the median (WT, n=7; KO, n=7). (C) The distance of CFSElabeled LSK cells from the endosteum in the midsection or the trabecular regions of the femur in a lodgment assay. Each dot represents an individual cell (midsection WT: n=20; midsection KO: n=10; trabecular WT: n=22; trabecular KO: n=12). *p < 0.05
sc-12-0678_sm_SupplFigure4b.tif391KSupplementary Figure 4. Alcam- deficient HSCs are able to home to BM and localize to endosteum. (A) Representative FACS plot for gating eFour670- labeled test cells and Violet- labeled input control cells. (B) Relative amount of eFour670- labeled Alcam−/− (KO) or WT LSK cells detected in BM, spleen (SP), peripheral blood (PB) and liver (LV) 4 hours after transplantation. Data was normalized with co- injected input control cells stained with CellTrace Violet (Invitrogen). Each dot represent an individual mouse, lines indicate the median (WT, n=7; KO, n=7). (C) The distance of CFSElabeled LSK cells from the endosteum in the midsection or the trabecular regions of the femur in a lodgment assay. Each dot represents an individual cell (midsection WT: n=20; midsection KO: n=10; trabecular WT: n=22; trabecular KO: n=12). *p < 0.05
sc-12-0678_sm_SupplFigure5a.tif38KSupplementary Figure 5. Alcam- deficiency does not alter G- CSF induced HSC mobilization. Alcam−/− or WT mice were subcutaneously injected with G- CSF JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal (300ug/ kg) for 5 consecutive days and phenotypic HSC subsets were analyzed by flow cytometry 6 hours after the last injection. Frequencies of phenotypic LT- HSC and STHSC in the bone marrow (A), spleen (SP) (B) and peripheral blood(PB) (C) determined by FACS. (D) The number of CFU- C in 1,000 mobilized PB cells. Each dot represent an individual mice, lines indicate the median (n=5).
sc-12-0678_sm_SupplFigure5b.tif34KSupplementary Figure 5. Alcam- deficiency does not alter G- CSF induced HSC mobilization. Alcam−/− or WT mice were subcutaneously injected with G- CSF JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal (300ug/ kg) for 5 consecutive days and phenotypic HSC subsets were analyzed by flow cytometry 6 hours after the last injection. Frequencies of phenotypic LT- HSC and STHSC in the bone marrow (A), spleen (SP) (B) and peripheral blood(PB) (C) determined by FACS. (D) The number of CFU- C in 1,000 mobilized PB cells. Each dot represent an individual mice, lines indicate the median (n=5).
sc-12-0678_sm_SupplFigure6a.tif47KSupplementary Figure 6. Hematopoietic profile of 12- month old mice. Red blood cell (RBC) (A), hematocrit (HCT)(B), Hemoglobin (Hb)(C), and platelets (PLT) counts (D). (E) Frequencies of peripheral B- T- and myeloid cells in the blood determined by FACS. Each dot represents an individual mice, lines indicate the median (WT: n=11; KO: n=14). (F) Frequencies of B- T- and myeloid cells in the bone marrow determined by FACS. (WT: n=5; KO: n=4)
sc-12-0678_sm_SupplFigure6b.tif31KSupplementary Figure 6. Hematopoietic profile of 12- month old mice. Red blood cell (RBC) (A), hematocrit (HCT)(B), Hemoglobin (Hb)(C), and platelets (PLT) counts (D). (E) Frequencies of peripheral B- T- and myeloid cells in the blood determined by FACS. Each dot represents an individual mice, lines indicate the median (WT: n=11; KO: n=14). (F) Frequencies of B- T- and myeloid cells in the bone marrow determined by FACS. (WT: n=5; KO: n=4)
sc-12-0678_sm_SupplTable1.tif752KSupplementary Table 1
sc-12-0678_sm_SupplTable2.tif60KSupplementary Table 1

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