Fig 2: The ee-As4S4 induced multi-lineage differentiation in vivo. (A) Illustration of the bone marrow assay and the gated scheme, cells in Gate 1 are leukemia cells. (B–D) Overlap distribution of CD11b, Ter119, and CD41 expression in the leukemia cells in the control group and the ee-As4S4-treated group. (E–G) Mean fluorescence intensity of the three markers in leukemia cells. (n=4) (H–J) Immunofluorescence staining of the three markers in femurs of mice from control and ee-As4S4 group.

Fig 3: Kupffer cell CLEC4F is a dominant receptor in mediating the rapid clearance of desialylated platelets in the liver.A Flow cytometry analysis of galactose exposure (RCA I binding) on platelets of either WT or Clec4f-/- recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. MFI, Mean fluorescence intensity. B Percentage of platelets of either WT or Clec4f-/- recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. Platelet count was measured using flow cytometric analysis of peripheral blood based on anti-CD41. C Representative immunofluorescent images showing association of platelets (CD41, red) with Kupffer cells (F4/80, blue) in the liver sections. Endothelium, (CD31, green). D, E Quantification of association of platelets with liver Kupffer cells or Kupffer cell (KC) number at 2 h after a neuraminidase injection. n = 5–10 images/10 × fields/experiment. Data represent means ± SEM from three experiments. *P < 0.05.

Fig 4: Conditioned medium of megakaryocytes promoted T24 cell survival. K562 cells were treated with various concentrations of phorbol 12-myristate 13-acetate (PMA) (0–10 nM) for 4 days. (A) Proteins were extracted and subjected to Western blot analysis with indicated antibodies. Representative blots of four independent experiments are shown. The Western blots have been shown in Figure S1. (B) Cells were harvested and subjected to flowcytometric analysis for the measurement of surface presentation of CD41. (C) Cells were pelleted by centrifugation, spread onto glass slides, and stained with Wright–Giemsa dye. Scale bar: 300 µm. K562 cells were treated with PMA (0 and 10 nM) in the absence or presence of dipyridamole (10 µM), aspirin (100 µg/mL), or cytochalasin D (1 µg/mL) for 4 days. (D) Cells were harvested and subjected to flowcytometric analysis for the measurement of surface presentation of CD41. Bar graphs show relative genomic mean intensity of CD41-PE fluorescence. (E) The cultured media were harvested and centrifuged, and the resultant supernatants were named conditioned medium (CM). Equal amounts of CM and fresh medium were mixed and added to T24 cells (96-well) for 48 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay. (F) Equal amounts of CM obtained from PMA treatments (0 and 10 nM) and fresh medium were mixed and added to T24 cells (96-well) in the absence or presence of cytochalasin D (1 µg/mL) for 48 h. Cell viability was measured by the MTS reduction assay. * p < 0.05 vs. untreated group and # p < 0.05 vs. PMA (10 nM) group or K562/PMA (10 nM) CM group, n = 4.

Fig 5: Lack of ß3-AR and Nitric Oxide Synthase 1 (Nos1) Signaling in the Microenvironment Accelerates Aging(A, A', and A?) Frequency of CD150lo/-CD41- lymphoid-biased HSCs in endosteal BM CD34-LSK cells from the following mice: (A) young WT (n = 7), young Adrb3-/- (n = 7), or old Adrb3-/- (n = 5) mice; (A') lethally irradiated WT recipients of WT (n = 5) or Adrb3-/- (n = 5) BM cells; and (A?) lethally irradiated WT (n = 4) or Adrb3-/- (n = 3) recipients of WT BM cells.(B) Nitrate concentration in BM extracellular fluid (BMECF) of young WT (n = 6) or Adrb3-/- (n = 7) mice.(C) Number of lin-sca-1+c-kit+CD34-CD48-CD150+Vwf-eGFP- lymphoid-biased HSCs in primary BM culture from Vwf-eGFP mice treated with ß3-AR agonist (BRL37344, 10 µM), Nos1 inhibitor (L-VINO, 100 µM), or vehicle for 4 days (n = 8).(D–G) Representative immunofluorescence (D–F) and quantification (G) of c-kit+ (green) CD41+ (red) myeloid or megakaryocyte progenitors (arrowheads) in endosteal BM of WT (n = 10), Adrb3-/- (n = 8), or Nos1-/- (n = 4) mice. Endosteal BM is considered as regions within one-fifth marrow width from the bone surface. Scale bar, 300 µm.(H–K) Representative immunofluorescence (H-J) and quantification (K) of CD41+ (red) megakaryocytes adjacent (arrowheads) or nonadjacent (asterisks) to EMCN+ (green) BM vasculature of WT (n = 9), Adrb3-/- (n = 6), or Nos1-/- (n = 4) mice. Scale bar, 250 µm.(L–N') Representative whole-mount immunofluorescence of CD31 (green) and EMCN (red) in WT (n = 7), Adrb3-/- (n = 5), or Nos1-/- (n = 4) BM. Arrowheads in insets (L'), (M'), and (N') depict CD31hiEMCN- capillaries.(O and P) Quantification of (O) CD31hiEMCNhi transition zone vessels and (P) CD31hiEMCN- capillaries with <6 µm diameter. Scale bar, 250 µm. Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (A' and A?) Unpaired two-tailed t test. (A–C, G, K, O, and P) One-way ANOVA and Bonferroni pairwise comparisons.