Human B, T, and HK cells were prepared from human tonsils obtained from children undergoing tonsillectomy at Asan Medical Center (Seoul, Korea). This study was approved by the Institutional Review Board of Asan Medical Center (Approval number, 2012-0636; approval date, August 31, 2012). Tonsillar B cells were prepared as previously described (9). Briefly, tonsillar mononuclear cells were subjected to 2 sessions of T cell depletion by rosetting with SRBC; the resulting cells contained more than 98% CD20⁺ cells as analyzed by FACScan (Becton Dickinson, Sunnyvale, CA, USA). T cells were isolated from the rosette-forming cell pellets after lysing SRBC; the resulting cells contained more than 95% CD3⁺ cells. HK cells were primary cells obtained from human tonsils and were used until they displayed degenerate features in culture. They were prepared as described by Kim et al. (10) and maintained in RPMI-1640 (Irvine Scientific, Santa Ana, CA, USA) containing 10% fetal calf serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin G (Sigma-Aldrich, St. Louis, MO, USA), and 100 µg/ml streptomycin (Invitrogen). The purity and phenotype of typical HK cells are presented elsewhere (11).

Cryostat sections of human tonsils were fixed in cold acetone for 10 min. The sections were rehydrated in PBS and blocked for 10 min with Protein block (Dako Korea Ltd, Seoul, Korea). The slides were double-stained first with anti-syntenin antibody (IgG1; Santa Cruz Biotechnology, Paso Robles, CA) at 4℃ for 12 h. After wash, goat anti-mouse IgG1-FITC (Jackson Immunoresearch, West Grove, PA, USA) was added, followed by incubation with PE-labeled anti-CD19, anti-CD3 antibodies (Becton Dickinson), or unconjugated CNA.42 (IgM; Abcam, Cambridge, UK). CNA.42 was visualized with goat anti-mouse IgM-PE. The coverslips were mounted onto slides using Fluorescent-mounting media (Dako). The relative positional distribution of the 2 fluorochromes was visualized and scanned using a confocal laser microscope (Fluoview FV300, Olympus, Tokyo, Japan). HK cells cultured in chamber slides were fixed with 4% formaldehyde for 10 min and incubated with anti-syntenin antibody for 1 h at room temperature. After wash, the slides were incubated with FITC-conjugated goat anti-mouse IgG1 and DAPI for nuclear staining.

Confluent HK cells were harvested by scraping, followed by the addition of cold PBS for centrifugation at 4℃, 500 g for 1 min. After discarding the supernatants, 100µl of a mixture containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 0.5 mM leupeptin, and 10% NP-40 was added before centrifugation at 4℃, 17000 g for 5 min. The supernatants were taken as the cytoplasmic fraction. The nuclear fraction of pellets were lysed in PRO-PREP protein extraction solution (iNTRON, Seongnam, Korea).

Immunoblotting was carried out as previously described (12). Used antibodies were against syntenin, FAK, phosphorylated FAK (Cell Signaling Technology, Danvers, MA, USA), β-actin (Sigma-Aldrich), HRP-conjugated anti-mouse IgG (Jackson Immunoresearch), and HRP-conjugated anti-rabbit IgG (KOMA Biotech, Seoul, Korea). The membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (PIERCE, Rockford, IL, USA) and exposed to X-ray films. Densitometry was carried out on the blots by using the LabWorks image acquisition and analysis software (UVP, Upland, CA, USA).

HK cells were cultured to 50~60% confluence in 100-mm plates. For each plate, 40 nM of each siRNA (Ambion Inc, Austin, TX, USA) and 24µl of Lipofectamine™ (Invitrogen) were separately diluted in 400µl of serum-free medium without antibiotics, mixed together, and incubated at room temperature for 45 min. The sequences of syntenin siRNA duplexes used; control (Neg-siRNA#2, sequence not disclosed by Ambion); sense (5'-GCACCAAGCAUUAUGAAAATT-3'), antisense (5'-UUUUCAUAAUGCUUGGUGCCA-3'). The plates were then washed with serum-free medium, added with 5 ml serum-free medium, and then with the diluted solutions. The plates were incubated at 37℃ for 8 h, followed by the addition of a growth medium containing 10% serum. After 48 h of additional incubation, cells were used in the experiments. The degree of gene-silencing was assayed by immunoblotting.

Purified B, T, and HK cells were stained with anti-syntenin antibody and then with goat anti-mouse IgG-FITC after membrane permeabilization with saponin. Flow cytometric analysis was carried out on a FACSCalibur with CELLQuest software (Becton Dickinson).

In order to examine whether human lymphoid tissue would express syntenin, we conducted immunohistochemical analysis with frozen tonsil sections. The sections were dual-stained with antibody against syntenin and antibodies against CD3, CD19, or CNA.42 specific for T cells, B cells, or FDCs, respectively, followed by confocal microscopic observation. Syntenin was expressed diffusely throughout the tissue but denser inside GCs than in the extrafollicular area (Fig. 1). Only small fractions of T cells expressed syntenin as determined by occasional appearance of CD3 and syntenin co-localized cells (Fig. 1A). The co-localization of syntenin with CD19 was more frequent than that with CD3 (Fig. 1B), implying that a significant fraction of B cells expresses syntenin. However, inasmuch as anti-CD19 antibody is also reactive with FDC (13) only small subsets of B cells appear to express syntenin. Most FDCs expressed syntenin as concluded by the intensive overlapping of CNA.42 and syntenin (Fig. 1C). Control sections that were stained with isotype-matched antibodies did not exhibit noticeable binding to antibodies (data not shown). These in situ data prompted us to explore syntenin expression with isolated cells from the tonsil; B, T, and HK cells. HK cells were used due to the paucity of bona fide FDCs obtainable from a tonsil. The expression of syntenin in T and B cells was confirmed by immunoblotting and flow cytometry. Only a small fraction of T and B cells expressed syntenin (Fig. 2). However, syntenin was strongly expressed in HK cells, in line with confocal microscopic findings in situ. At subcellular levels, most syntenin molecules were localized in the cell membrane, nuclear membrane, and cytoplasm (Fig. 3A). Syntenin was not expressed in the nuclei of HK cells except the nuclear membrane, and a significant overlapping of syntenin and DAPI was not observed. These data obtained by confocal microscopy were confirmed by immunoblotting. Syntenin molecules were largely restricted to the cytoplasmic fraction (Fig. 3B).

Given that HK cells express syntenin strongly in the cell membrane (Fig. 3A), we investigated its role in adhesion of HK cells to tissue-culture treated plastics. This reaction mimics focal adhesion (FA) that is a close contact of adherent cells with extracellular matrices (ECM), a dynamic process requiring coordinated rearrangement of cytoskeletons. The siRNA technology was adopted to knock-down syntenin molecules in HK cells (Fig. 4A). HK cells adhered to plastics in a time-dependent fashion, which correlated with FAK phosphorylation (Fig. 4B). The phosphorylation peaked 2 h post adhesion, and then remained at the similar level. In contrast, FAK phosphorylation kinetics were markedly impaired when syntenin was knocked down. The phosphorylation level obtained after 4 h of incubation was only comparable to that observed 1 h post-incubation in control HK cells. However, syntenin knockdown did not change cell morphology or delay plastic adhesion (data not shown), implying that plastic adhesion of HK cells is a complicated process involving many molecules in addition to syntenin. These results suggest that syntenin takes part in the FA of HK cells by promoting phosphorylation of FAK molecules.