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Fig. 4. Induction of epimorphin secretion in stable transfectants. (A) Appearance of a 34 kDa full-length epimorphin at cell surface (Ex) and a 30 kDa form in the supernatant (Sup) of PT67 cells stably expressing the TE transgene following treatment with subtoxic levels of cycloheximide (CHX), actinomycin D (Acti-D), camptothecin (Camp), or the Ca²⁺ ionophore A23187. Treatment with DMSO (–) or the Ca²⁺-chelator EDTA did not lead to membrane translocation. (B) Epimorphin secretion is not a consequence of cell death. The calpain inhibitor Calpeptin decreased epimorphin secretion by partially inhibiting cycloheximide-dependent cell death. However, neither cell stresses (0.5 mM EDTA) nor temporal heat shock (42°C for 1.5 hours at day 2) resulted in a detectable increase in epimorphin secretion. Cell viability at day 3 was quantified with alamarBlue^TM assay with non-treated cells being treated as 100% viable. Data are the mean ± s.d., n=4, ^*P<0.05. (C) Epimorphin coprecipitates with annexin II and synaptotagmin1 (full-length 65 kDa synaptotagmin and the 40 kDa extravesicular domain) from lysate of the transfectants, regardless of cycloheximide treatment (upper panel). Epimorphin is secreted to medium in response to cycloheximide-treatment in a secretory complex with annexin II and the extravesicular domain of synaptotagmin, both of which directly bind to phosphatidylserine (middle panel). Cycloheximide induces phosphatidylserine externalization as judged by binding of FITC-labeled annexinV (Biovision) to the non-permeabilized cells (lower panel). Cell nuclei were visualized with propidium iodide (PI) after permeabilization. Bar, 25 µm. (D) Anti-epimorphin antibodies (red) bind to the surface of non-permeabilized PT67 cells expressing TE transgene following treatment with CHX, whereas binding of anti-β-actin antibodies (green) requires cell permeabilization. Bar, 10 µm.

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