Fig 2: Gating Strategy for Sorting HSC(A) Gating strategy for sorting CD150+CD41-CD48-Flt3-CD34- LSK cells.(B) Gating strategy for sorting CD150+CD48-Flt3-EPCR+ LSK cells

Fig 3: Gal-3 is highly expressed by LT-HSCs in the vascular niche.a Relative expression level of Gal-3 mRNA in LT-HSC, ST-HSC, MPP2, MPP3, and MPP4 cells in the BM of WT mice (LT-HSC: CD150+CD48-Flt3–LSK; ST-HSC: CD150-CD48-Flt3-LSK; MPP2: CD150+CD48+Flt3-LSK; MPP3: CD150-CD48+Flt3-LSK; MPP4: CD150-CD48+Flt3+LSK). n = 4 biological replicates, compared by two-sided t test. Data are presented as mean values ± S.D. b Detection of Gal-3 protein (green) by immunofluorescence (IF) in LT-HSC, ST-HSC, MPP2, MPP3, and MPP4 populations in the BM of WT mice. DAPI (blue) was used to detect nuclei. A representative image is shown (n = 3 independent experiments with at 2–3 cell section per experiment). Scale bar, 50 µm. c Quantification of the IF analysis developed as described in b. For quantification, three cell sections (per each cell type) were analyzed (ten random fields per section) using Image J software (two-sided t test). Data are presented as mean values ± S.D.. Right-hand panels show the ratio between Gal-3 (green) and nuclear area (blue) which was calculated to assess the diversity of Gal-3 expression. d IF staining of Gal-3 (green), CD150 (blue), and Endomucin (red) in BM sections from WT mice. Endomucin was selected as an endothelial cell marker in BM and the dashed box indicates areas shown at higher magnification. A representative image is shown (n = 4 biological replicates). Scale bars, 100 µm and 50 µm (inset). e Distances between Gal-3+ HSC (Gal-3+CD150+) or Gal-3- HSC (Gal-3-CD150+) and blood vessels (Endomucin+) in the metaphysis and diaphysis area of WT BM. n = 4 biological replicates (six random fields per BM sections), compared by two-sided t test. Data are presented as mean values ± S.D. f IF staining of Gal-3 (green), CD150 (blue), and Endomucin (red) in BM sections from WT mice at different time points after 5-FU treatment. Endomucin was selected as above. A representative image is shown (n = 4 biological replicates). Scale bar, 100 µm. g Distances between Gal-3+ HSC (Gal3+CD150+) or Gal-3- HSC (Gal3-CD150+) and blood vessels (Endomucin+) in the metaphysis and diaphysis area of WT BM at different time points after 5-FU treatment. n = 4 biological replicates at each time point (six random fields per BM sections), compared by two-sided t test. Data are presented as mean values ± S.D.

Fig 4: Transcriptional regulation of Gal-3 expression mediated by Ang-1/Tie2 or Thpo/Mpl signaling through the PI3K/AKT-NFkB pathway.a–c Relative expression level Gal-3 mRNA in LT-HSCs (CD150+CD48-Flt3-LSK) in BM of WT mice after stimulation with Ang-1 (a), Thpo (b), and CXCL12 (c) (n = 4 biological replicates, compared by two-sided t test). Data are presented as mean values ± S.D. and n.s. not significant. d Experimental schema for ex vivo inhibition of the PI3K/AKT or MEK/ERK pathway. e, f Analysis of Tie2 and Mpl downstream signaling pathways for the expression of Gal-3. WT LT-HSCs (CD150+CD48-Flt3-LSK) were preincubated with either PD98059 (50 µM, MEK inhibitor), LY294002 (10 µM, PI3K inhibitor), or DMSO (control, 0.05%) for 30 min before Ang-1 (250 ng/ml, e) or Thpo (100 ng/ml, f) stimulation for 8 h. The samples were analyzed by real-time PCR (n = 3 biological replicates, compared by two-sided t test). Data are presented as mean values ± S.D. and n.s. not significant. g (Left) Representative flow cytometry histogram showing changes in AKT phosphorylation between Ang-1- or Thpo-treated and PBS-treated LT-HSCs (CD150+CD48-Flt3-LSK). (Right) Quantification of AKT phosphorylation expressed as arbitrary units of the mean fluorescence intensity (MFI a.u.) in LT-HSCs after stimulation with Ang-1 or Thpo (n = 3 biological replicates, compared by two-sided t test). Data are presented as mean values ± S.D. h, i Nuclear translocation of p65 (NF-?B) in response to Tie2 or Mpl activation. LT-HSCs (CD150+CD48-Flt3-LSK) were treated with Ang-1 (h) or Thpo (i) and stained with anti-p65 (red) and DAPI (blue) antibodies. A representative image is shown (n = 3 biological replicates). Scale bar, 50 µm. j Experimental schema for ex vivo BAY-11-7082 (NF-?B inhibitor) treatment. k, l Relative expression level of Gal-3 mRNA in WT LT-HSCs (CD150+CD48–Flt3-LSK) on ex vivo BAY-11-7082 treatment. LT-HSCs were preincubated with BAY-11-7082 or DMSO (0.05%) for 30 min before Ang-1 (250 ng/ml, k) or Thpo (100 ng/ml, l) stimulation for 8 h (n = 3 biological replicates, compared by two-sided t test). Data are presented as mean values ± S.D. and n.s. not significant. m Experimental schema for in vivo BAY-11-7082 treatment. n (Left) Representative flow cytometric analysis of LT-HSC (CD150+CD48-Flt3-LSK) frequencies (red box) in BM of PBS-treated (Ctrl) or BAY-11-7082-treated (BAY) mice. (Right) Bar graph showing the percentage of LT-HSCs in the Flt3-LSK population (n = 4 biological replicates). o Relative expression level Gal-3 and p21 mRNA in LT-HSCs (CD150+CD48-Flt3-LSK) in BM of PBS-treated (Ctrl) or BAY-11-7082-treated (BAY) mice. Cells were harvested at 12 h (n = 4 biological replicates, compared by two-sided t test). Data are presented as mean values ± S.D.

Fig 5: Administration of ß-Glucan Drives Expansion of HSPC SubpopulationsWT mice were injected with ß-glucan or PBS, and BM analysis was performed after 24 hr.(A) Representative fluorescence-activated cell sorting (FACS) plots for the identification of hematopoietic progenitor cells. After gating for Lin- cells, LSK cells were characterized as cKit+Sca1+ cells. LSK cells subpopulations were further characterized as MPP (CD48+CD150-LSK), ST-HSC (CD48-CD150-LSK), and LT-HSC (CD48-CD150+LSK).(B and C) Cell numbers of LSKs, MPPs, ST-HSCs, and LT-HSCs (B) and cell percentages of the same populations in total BM cells (C) of mice at 24 hr after the administration of PBS or ß-glucan (ns = 11 and 12 mice).(D) Representative FACS plots for the identification of MPP subpopulations. After gating for LSK cells, MPP4 cells are characterized as CD48+Flt3+CD150-LSK, MPP3 cells are characterized as CD48+Flt3-CD150-LSK, and MPP2 cells are characterized as CD48+Flt3-CD150+LSK.(E) Frequency of MPP subpopulations in LSK cells in the BM of mice at 24 hr after the administration of PBS or ß-glucan (n = 5 mice per group).(F and G) Representative FACS plots for the identification of CD41+ LT-HSCs (F) and frequency of CD41+ LT-HSCs (in total LT-HSCs) (G) in the BM of mice at 24 hr after the administration of PBS or ß-glucan (n = 5 mice per group).Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.See also Figure S1.