Fig 1: Flow cytometer analysis of endothelial adhesion molecules, (A) VCAM-1 and (B) ICAM-1, expression in HUVEC incubated with vehicle CuSO4 (10 µmol/L) + 0.1 mmol/L EDTA (gray bar), Nat Alb (white bars), or Ox Alb (black bars).Notes: HUVEC were treated with different doses (mg/mL) of native and oxidized albumin for 24 hours. MFI are presented as mean ± SD, n=3, in duplicate. *P<0.05 versus vehicle; #P<0.05 versus at all Nat Alb doses.Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; MFI, median fluorescence intensity; HUVECs, human umbilical vein endothelial cells; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; EDTA, ethylenediaminetetraacetic acid; SD, standard deviation.
Fig 2: Effect of CD146 expression in stroma on BrdU uptake by cocultured CD34+ hematopoietic cells in vitro, and on engraftment of CD34+ MDS marrow cells in NSG mice in vivo. (a) Flow cytometric analysis showed prominent expression of CD146 on HS27a, but not on HS5 stroma. Knockdown (KD) of CD146 in HS27a cells using four different siRNAs (no. 1–4 in comparison with a scrambled siRNA) reduced CD146 expression to levels comparable to those in HS5 cells. Conversely, overexpression of CD146 (over-CD146) in HS5 cells using a lentiviral construct (pLOC vector, over-CD146, in comparison to a scrambled vector (SCR)) increased CD146 expression to levels comparable to those in unmodified HS27a cells (see also Supplementary Figure S4b; upper row). (b) BrdU uptake by hematopoietic cells after coculture with either unmodified HS27a stroma wild type (WT), HS27a cells with KD of CD146 (146 KD), or HS5 cells overexpressing CD146 (over-CD146), in comparison with unmodified (WT) HS5 cells. BrdU uptake was highest in KG1a cells, followed by MDS-derived CD34+ cells and CD34+ cells from healthy donors. Results of coculture with HS27a cells with KD of CD146 approached those with unmodified HS5 cells, whereas, conversely, BrdU uptake in coculture with CD146 overexpressing HS5 cells did not differ significantly from that in coculture with unmodified HS27a cells (Student's t-test; mean±s.e.m. of three experiments ). (c) Engraftment of CD45+ marrow cells from two patients with RAEB-2 and RAEB-1, respectively, coinjected with unmodified HS5 stroma (HS5), unmodified HS27a stroma (HS27a) or HS5 cells overexpressing CD146 (HS5-CD146), in marrow and spleen of NSG mice, determined at 5–7 weeks after transplantation. The table shows, in addition, the proportions of human clonal and non-clonal CD34+cells (from patient 23) in mouse marrows and spleens after coinjection with HS27a and HS5-CD146 cells (day 35), respectively. Additional data on FISH and flow cytometric analysis, as well as immunohistochemical analysis of ICAM1 and CD146 expression are shown in Supplementary Figure S4. RAEB-1 or 2, refractory anemia with excess blasts 1 or 2, respectively; NBM, normal bone marrow.
Fig 3: Localization and colocalization of human HS27a stroma and hematopoietic cells in murine marrow (BM) and spleen. (a) Confocal microscopy showing colocalization of KG1a cells and HS27a stroma in fresh frozen sections of spleen (original magnification: × 40): HS27a cells are labeled with FITC-conjugated anti-human ICAM1 antibody. Red indicates human CD45+ KG1a cells, whereas blue shows nuclear staining with DAPI (both murine and human nuclei stain with DAPI). The right lower panel represents the merged picture. Superimposition of the FITC signals of the anti-ICAM1 antibody and the Alexa 647 signals of the CD45 antibody results in a yellow hue of signals. White arrows indicate examples of staining for colocalizing HS27a stroma and CD45+ cells. (b) Spleen sections (formalin fixed) stained with anti-human ICAM1 antibody (green) and anti-CD45 antibody (red), merged in the right panel (original magnification= × 40). The lower panels show isotype controls. (c) Immunohistochemical determination of the distribution of primary MDS cells (labeled with anti-human (h) CD34 and CD45 antibodies) and HS27a stroma (labeled with anti-human ICAM1 antibody) in the bone marrow and spleen of NSG mice. White arrows indicate identical coordinates on sequential sections (section distance=4 µm; orignal magnification= × 40). (d) Immunohistochemical staining of formalin-fixed spleen sections labeled with anti-human CD146 antibody (original magnification= × 40). Dark brown identifies 3,3'-diaminobenzidine chromagen linked to the CD146 antibody, identifying HS27a cells; blue represents counter staining with hematoxylin. Samples from two mice injected with primary CD45+ MDS cells from two different patients. Each figure represents one example of 2–4 similar experiments. (e) Flow cytometric analysis of bone marrow (BM) and spleen cells harvested from mice transplanted with human MDS marrow without coinjection of stroma (without stroma) or coinjected with unmodified HS5 or HS27a stroma. Cells positive for human ICAM1and CD146 (typical for HS27a stroma; see also Figure 4a and Supplementary Figure S4b) were identified in marrow and spleen from mice injected with HS27a.
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