Fig. 3

NOXA activated the mitochondria-mediated apoptosis pathway in response to the combination treatment by regulating MCL1. A, B siNC-, siTP53-, or siATF3-transfected A549 tumor spheroids were treated with individual or combination treatments of cisplatin and narciclasine. A Cell viability was assessed after treatment for 48 h. *p < 0.05 versus vehicle-treated siNC; $p < 0.05 versus narciclasine-treated siNC; †p < 0.05 versus combination-treated siNC. Data are mean ± SEM from three independent experiments in triplicate. B The levels of the indicated proteins were assessed by western blotting after treatment for 24 h. Vinculin was used as the loading control. Data represent one of three independent experiments with similar results. C Twenty-four hours after the indicated treatment, mRNA levels of NOXA, TP53, and ATF3 were assessed using RT-qPCR. Data are presented as fold change in gene expression, normalized to GAPDH expression. *p < 0.05. Data are mean ± SEM from three independent experiments in triplicate. D, E siNC- or siNOXA-transfected A549 tumor spheroids were treated with individual or combination treatments of cisplatin and narciclasine for 48 h. D The expression of intrinsic apoptosis-related proteins was assessed by western blotting. Data represent one of three independent experiments with similar results. E Apoptosis was assessed using a cell death detection ELISA kit capable of detecting mono- and oligonucleosomes. *p < 0.05. Data are mean ± SD from three independent experiments in triplicate. F–H A549 tumor spheroids stably transfected with flag-tagged vector control or flag-tagged MCL1 were treated with individual or combination treatments of cisplatin and narciclasine for 48 h. Images were taken prior to viability assay. Scale bar: 100 μm. F Cell viability assay, G ELISA, and H western blotting were performed. *p < 0.05. Data in F and G are mean ± SEM and from three independent experiments in triplicate. Data in H represent one of three independent experiments with similar results