Fig. 5: Combination treatment with the two ALK inhibitors and a p53 activator induces activation of the intrinsic apoptosis pathway. | Cell Death Discovery

Fig. 5: Combination treatment with the two ALK inhibitors and a p53 activator induces activation of the intrinsic apoptosis pathway.

From: The p53 activator overcomes resistance to ALK inhibitors by regulating p53-target selectivity in ALK-driven neuroblastomas

Fig. 5

a,b Activation of mitochondria-mediated apoptotic pathway by combination treatment of the two ALK inhibitors with the p53 activator Nutlin-3. NB39-nu or NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. The activation of the caspase cascade as a result of combination treatment was detected by immunoblot analysis (a). The expression levels of caspases-8 and -9 in NB39-nu or NB1 cells are shown in (b). c The increase in cytosolic cytochrome c following combination treatment. NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. Cytosolic fractions were prepared by subcellular fractionation and the amount of cytochrome c present was quantified by ELISA. Data show the mean ± SEM (n = 3). *p < 0.05. d–g The caspase inhibitor Z-VAD-FMK, but not the RIPK1 inhibitor necrostatin-1, abolished the cell death elicited by the combination treatment. Z-VAD-FMK (100 µM) or necrostatin-1 (nec-1, 50 µM) were combined with the ALK inhibitors and Nutlin-3 as shown and a CytoTox GLO assay (d,e) or necroptosis assay (f,g) were performed. NB39-nu (d,f) and NB1 cells (e,g) were shown. Data indicate mean ± SD (n = 3). *p < 0.05. All experiments were repeated at least three times

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