Non-classical export of epimorphin and its adhesion to v-integrin in regulation of epithelial morphogenesis

JCS006247 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Secreted epimorphin lacks the TM domain and part of the SNARE domain. T7-tagged epimorphin was transfected into PT67 cells, and the transgene products were collected with antibodies against the N-terminus or C-terminus (anti-N or anti-C, respectively) of epimorphin and analyzed by western blotting with anti-T7 monoclonal antibody. anti-N, polyclonal antibodies affinity-purified with r-Epm (aa 1-188); anti-C, rat antiserum raised against synthetic peptide corresponding to C-terminus of epimorphin SNARE (aa 248-265, KEETKKAIKYQSKARRKK).

• Supplemental Figure S2 -

  Fig. S2. SCp2 cell adhesion to epimorphin is mediated by αv- and β1- integrins. SCp2 cells were seeded in dishes coated with biotinylated form of r-Epm or r-GFP and incubated 4 hours. The lysate of the whole culture was then immunoprecipitated with anti-integrin antibodies, which recognize the cytoplasmic domain of both human and mouse integrins. Antibodies against αv- and β1-integrins, but not α5- and β3-integrins, co-precipitate r-Epm (biotin r-EPM) but not r-GFP (biotin r-GFP). The substrate (r-Epm or r-GFP) retrieved with integrins was visualized with HRP- streptavidin.

• Supplemental Figure S3 -

  Fig. S3. Comparison of elements for non-classical secretion of epimorphin, IL-1 and FGF-1. (A) Stimuli for the extracellular secretion of these proteins that lack signal peptides. Several stress inducers lead to secretion of these proteins that lack signal peptides but their effect is not always the same for all proteins. Act-D; actinomycin D, CHX; cycloheximide, A23187; calcium ionophore. (B) Identified components in the secretion complex of epimorphin, IL-1a and FGF-1. *; proteins that can directly associate with phosphatidylserine

• Supplemental Figure S4 -

  Fig. S4. Expression profile of untagged and signal peptide-tagged epimorphin. Full-length epimorphin or epimorphin fused to the IL-2 signal peptide at the N-terminus was transfected into PT67 cells. The transgene products were analyzed with rabbit and rat anti-epimorphin antibodies. Note that untagged 34 kDa epimorphin is secreted but not the glycosylated 37 kDa form.

• Supplemental Figure S5 -

  Fig. S5. Epimorphin confers apoptosis resistance. Recombinant epimorphin (r-Epm), but not recombinant GFP (r-GFP), prevents starvation-induced cell death in HMECs. HMECs maintained in basal medium (not suplemented with pituitary extract, hydrocortisone, EGF and insulin) showed healthy morphology in the presence of r-Epm (10 μg/ml) even after 4 weeks. Neither PBS nor r-GFP (10 μg/ml) protect cell death. Micrographs show phase-contrast images after 4 weeks (left) and staining with hematoxilin (right). Bars, 100 μm.