Fig. 6

CD271 cleavage is required to activate A?^(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 A?^(25?35) at different time points. C, M121224 cells were seeded in six-well plates and treated with A?^(25?35) 40?mol/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 A?^(25?35) 40 ?mol/L for 23 hours, then MG132 (20 ?mol/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 A?^(25?35) 40 ?mol/L was added in the medium for 30 minutes and PI staining was performed CD271 + and ? sorted cells were treated with A?^(25?35) ± DAPT (300 nmol/L)/MG132 (20 ?mol/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 A?^(25?35) (40 ?mol/L) and stained with PI for cell-cycle evaluation by FACS.