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Fig. 7
Melanoma cell death is triggered by A?(25?35)-mediated activation of CD271?JNK pathway and mitochondrial ROS production. A, Western blot on melanoma cells were treated with A?(25?35) (40 ?mol/L) at different time points. CD271 and GAPDH belong to the same Western blot shown in Fig. 6B. B and C, M121224 wt cells were treated with A?(25?35) (40 ?mol/L) ± DAPT (200 nmol/L) or JNKi (SP600125; 300 nmol/L) and Western blot was performed. D, M121224 wt and CD271 KO cells were treated for 48 hours with A?(25?35) (40 ?mol/L). Lysates were pulled-down with CD271 Ab, and supernatant (Sup) and immunoprecipitates (IP) were immunoblotted with different Abs. E, Melanoma cells were treated with A?(25?35) (40 ?mol/L) ± JNKi (SP600125; 200 nmol/L) for 1 hour and stained with PI. The percentage of cell death was evaluated by FACS. Two-way ANOVA was used for statistical analysis. **, P < 0.01; ***, P < 0.001.F, Tumor slices were treated for 5 days and stained with Ki67 and S100 Abs. Ki67+ cells were quantified by QuPath. The average of 10 areas was normalized to the total S100. Two-way ANOVA was used for statistical analysis. ****, P < 0.00001. Scale bar, 100 ?m. ?Cntr? are the same pictures shown also in Fig. 3 (patient 2 and 3). G, Cells were stained with MitoSOX (5 ?mol/L) and the levels of mROS were measured by FACS. Data represent the mean ± SD of triplicate determinations. One-way ANOVA was used for statistical analysis. **, P < 0.01; ***, P < 0.001. H, Melanoma cells were treated with A?(25?35) (40 ?mol/L) ± NAC (5 mmol/L) for 72 hours and stained with MitoSOX. mROS were measured by FACS. I, M121224 were treated with A?(25?35) alone or in combination with JNKi (200 nmol/L) ± NAC (5 mmol/L). Cells were stained with PI and the percentage of cell death was evaluated by FACS. One-way ANOVA was used for statistical analysis. *, P < 0.05; ***, P < 0.001; ****, P < 0.00001. J, M121224 cells were treated with A?(25?35) and mROS were measured by FACS at different time points. K, M121224 CD271+ cells were treated with A?(25?35) (40 ?mol/L) ± NAC (5 mmol/L) for 48 hours. CD271 levels were evaluated by Western blot. L, Graphical representation of CD271?JNK?ROS pathway induced following A?(25?35) treatment. ns, nonsignificant.