Syntenin selectively promotes IL-5–driven proliferation. (A) Stable polyclonal TF-1 cell lines were generated that ectopically express HA-tagged full-length syntenin, syntenin lacking its N-terminal domain (PDZ1+2), or EGFP as control. All lines were more than 95% EGFP positive. Viable cell numbers of 6-day cultures in either IL-5–supplemented (■) or GM-CSF–supplemented (□) medium are displayed. An average of 4 independent experiments is depicted (± SD). (B) Anti–HA-tagged Western blot indicating the presence of HA-tagged syntenin or PDZ1+2 in selected TF-1 cell–derived clones. Two clones per group are shown. (C) Expression of IL-5Rα for each subclone was analyzed by flow cytometry. Histograms of isotype-stained (black) or IL-5Rα–stained (white) cells are indicated. (D) Proliferative responses of selected clones to IL-5 or GM-CSF after 72 hours. Viable cells (live gate, propidium iodide negative) were counted by flow cytometry after culturing the cells for 3 days without cytokine, or after addition of IL-5 or GM-CSF. Mean of triplicate samples (± SD) is indicated for 2 control EGFP-clones, 2 clones transduced with syntenin, and 2 clones transduced with PDZ1+2. An average of 4 independent experiments is depicted (± SD). (E) Surface levels of clones expressing syntenin, PDZ1+2, or EGFP were incubated with IL-5 (10nM) for various time points and remaining surface expression was analyzed using flow cytometry. Data represent mean ± SD of triplicates.