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. 2005 Nov;79(21):13326–13337. doi: 10.1128/JVI.79.21.13326-13337.2005

FIG. 5.

FIG. 5.

Cell lineage and subcellular distribution of galectin 9. (a) Constitutive expression of galectin 9 in various types of lymphoid and epithelial cells. EBV1 and NAD+C15 are EBV-transformed B-cell lines (LCL). Ramos, BL2, and Daudi are Burkitt lymphoma cell lines. BL2-B95 was obtained by EBV conversion of BL2 cells infected with the B95-8 strain. Regarding epithelial cells, all protein extracts were prepared from xenografted tumors (C15 and C17 are permanently propagated into nude mice; other epithelial cells were specially grown as tumors into nude mice for the consistency of this analysis). For each cell type, 20 μg of whole-cell protein extract was analyzed by Western blotting. (b) Association of galectin 9 with membrane rafts in both LMP1-positive and -negative NPC cells. C15 and C17 tumor pieces were homogenized in MBS-Triton buffer, subjected to a raft flotation assay, and analyzed by Western blotting. The flotation assay generates three cell fractions in addition to the raft fraction: an LSP and an HSP, containing mostly cytoskeletal elements, and fraction F40 recovered in the 40% sucrose layer of the step gradient. F40 contains most components of the cytosol and nonraft membranes (11). Ten micrograms of protein was loaded per lane. In agreement with the data presented in panel a, galectin 9 was much more abundant in C15 than in C17 material (corresponding films were exposed 2 and 10 min, respectively). However, the ratio of galectin 9 concentration in the rafts compared to the initial extract was in the same range for the C15 (LMP1-positive) and the C17 (LMP1-negative) xenografts. (c) Galectin 9 expression in clinical specimens of NPC. NPC tissue sections from nine patients were stained with a primary anti-galectin 9 and a secondary peroxidase-labeled antibody and finally counterstained with Mayer's hematoxylin. Galectin 9 expression by malignant cells was found in all 9 biopsies; it was very intense for seven out of nine cases. Results from two patients (patients 2 and 3) are presented in this figure. A very strong expression of galectin 9 is visible in tumor cells (T). In contrast, galectin 9 is at a low abundance in the lymphoid stroma (LS) and the adjacent nonmalignant mucosa (PM; parakeratosic mucosa). In the case of patient 3, the staining of malignant cells is predominant at the plasma membrane.