Figure 2. CDON triggers apoptosis through CDON proteolytic cleavage and recruitment and activation of caspase-9.

(A) In vitro–translated CDON intracellular domain (CDON-IC) wild-type (wt) or mutated on one (left panel) or two (right panel) aspartic acid residues were incubated in the absence or in the presence of recombinant purified active caspase-3. Autoradiographs show the cleavage by caspase-3 of CDON-IC wt, whereas CDON-IC–D1153N was weakly cleaved and CDON-IC–D1153N–D1164N was almost completely resistant to cleavage. The band appearing in the CDON D1164N–D1153N probably represents a cryptic site. (B) Schematic representation of CDON and its different mutant constructs. CDON-main (D1153) and secondary (D1178) caspase cleavage sites are shown. (C–D) Apoptotic cell death induction as measured by caspase-3 activity (C) was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids and by TUNEL (D) staining was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids. Quantifications of TUNEL positive cells (orange) and nuclei (Hoechst in blue) are shown. (E) Apoptotic cell death induction as measured by caspase-3 activity was quantified in HEK293T cells transfected with constructs encoding full-length CDON or the CDON hypothetical fragments resulting from its cleavage by caspase at D1153 (CDON1–1153 and CDON 1154–1250). (F) HEK293T cells were transfected with constructs encoding CDON and/or caspase-9 and cell lysates were subjected to immunoprecipitation with a CDON-specific antibody. CDON and caspase-9 proteins were detected by Western blot in immunoprecipitated and input fractions. (G) Caspase-9 activity was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON (D1153N) expression plasmids. For (C–F) and (G), data are means of at least three independent assays. Error bars indicate s.d.