Fig. 5

NOXA regulation in response to ROS generated by combination treatment with cisplatin and narciclasine is mediated via the IRE1α–JNK/p38 axis. A549 tumor spheroids were pretreated with A 50 nM of GSK414, a PERK inhibitor, or B 100 nM of KIRA6, an IRE1α inhibitor, for 1 h, followed by treatment with 10 μM cisplatin, 0.3 μM narciclasine, or their combination for 4 or 24 h. NOXA mRNA levels were evaluated using RT-qPCR. Data are presented as fold change of NOXA expression, normalized to GAPDH expression. †p < 0.05 versus narciclasine; *p < 0.05 versus combination. Data are mean ± SEM from three independent experiments in triplicate. C Under the treatment condition described in A and B, levels of proteins involved in the UPR pathway were analyzed by western blotting. Data represent one of three independent experiments with similar results. D A549 tumor spheroids were pretreated with 10 μM of SP600125, a JNK inhibitor or SB203580, a p38 inhibitor for 1 h, followed by treatment with 10 μM cisplatin, 0.3 μM narciclasine, or their combination for 4 h or 24 h. NOXA mRNA levels were evaluated using RT-qPCR. Data are presented as fold change in NOXA expression, normalized to GAPDH expression. †p < 0.05 versus narciclasine; *p < 0.05 versus combination. Data are mean ± SEM from three independent experiments in triplicate. E Under the treatment condition described in D, levels of proteins involved in UPR pathway were analyzed by western blotting. Data represent one of three independent experiments with similar results. F A549 tumor spheroids were pretreated with 10 mM NAC (N-acetylcysteine) or 0.5 mM glutathione ethyl ester (GSH-EE) for 1 h, followed by treatment with 10 μM cisplatin, 0.3 μM narciclasine, or their combination for 1 or 24 h. Levels of proteins involved in the UPR pathway were analyzed by western blotting. Data represent one of three independent experiments with similar results