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Fig. 6
CD271 cleavage is required to activate Aβ(25–35)-induced apoptosis. A, Representation of CD271 cleavage sites and the inhibitors used. B, CD271 levels were evaluated by Western blot after treatment with Aβ(25–35) at different time points. C, M121224 cells were seeded in six-well plates and treated with Aβ(25–35) 40μmol/L ± DAPT (200 nmol/L) for 16 and 72 hours. Protein extracts were immunoblotted with CD271. D, 1205Lu CD271 EV and FL (full length) cells were pretreated with Aβ(25–35) 40 μmol/L for 23 hours, then MG132 (20 μmol/L) + DAPT (300 nmol/L) were added for 1 hour. CTF and ICD formation was evaluated by Western blot. E–G, 1205Lu FL cells were treated for 1 hour with DAPT (300 nmol/L; E), then Aβ(25–35) 40 μmol/L was added in the medium for 30 minutes and PI staining was performed CD271 + and − sorted cells were treated with Aβ(25–35) ± DAPT (300 nmol/L)/MG132 (20 μmol/L)/TAPI2 (500 nmol/L; F and G). CD271 was evaluated by Western blot. H, Cell cycle by FACS. Two-way ANOVA was used for statistical analysis. *, P < 0.05; ***, P < 0.001; ****, P < 0.00001. I, CD271 FL and ECD structure. J, 1205Lu wt, EV, FL, and ECD lysates were immunoblotted for CD271 Ab. K, Melanoma cells were treated for 1 hour with Aβ(25–35) (40 μmol/L) and stained with PI for cell-cycle evaluation by FACS.