Fig. 1

Narciclasine significantly enhanced the sensitivity of NSCLC tumor spheroids to cisplatin. A, B A549 tumor spheroids were treated with various concentrations of cisplatin (A) or narciclasine (B) for 72 h, and cell viability was determined by measuring cellular adenosine triphosphate (ATP) content. Data are mean ± SD from three independent experiments in triplicate. C The viability of tumor spheroids was assessed following cisplatin, narciclasine, or the combination treatment at the indicated concentrations for 72 h. *p < 0.05 versus same dose of cisplatin; †p < 0.05 versus same dose of narciclasine; $, less than half of cell viability compared with each treatment; ▼, selected combination dose. Data are mean ± SD from three independent experiments in triplicate. D Combination index (CI) values calculated using CompuSyn. CI of < 1.0 indicates synergism. E Tumor spheroids were treated with the vehicle, 10 μM cisplatin, 0.3 μM narciclasine, or both for the indicated time periods. Images were taken prior to viability assay. *p < 0.05 versus cisplatin; †p < 0.05 versus narciclasine. Data are mean ± SEM from three independent experiments in triplicate. Scale bar: 100 μm. F Whole-cell lysates were prepared from A549 tumor spheroids 48 h after treatment with cisplatin, narciclasine, or the combination. The levels of cleaved caspases were analyzed by western blotting. GAPDH was used as the loading control. Data represent one of three independent experiments with similar results. G Tumor spheroids were treated with cisplatin, narciclasine, or the combination for 48 h. Live cells were stained green with calcein-acetoxymethyl ester, whereas dead cells were stained red with ethidium homodimer-1. Images were taken at magnification of 100× using the Operetta high-content analysis system. Scale bar: 200 μm. Results of the quantitative analysis of the dead cell area are shown in the right panel. *p < 0.05. Data are mean ± SD from three independent experiments in triplicate. H Tumor spheroids were dissociated 48 h after the indicated treatments and analyzed using flow cytometry. Apoptotic cells were presented as percentages of annexin V-positive/7-amino-actinomycin D (7-AAD)-negative cells. *p < 0.05. Data are mean ± SEM from three independent experiments in triplicate. I A549 tumor spheroids were treated with cisplatin, narciclasine, or the combination for 48 h, and apoptosis was analyzed using a Cell Death Detection ELISA kit capable of detecting mono- and oligonucleosomes. *p < 0.05. Data are mean ± SEM from three independent experiments in triplicate. J Tumor volume and body weight were measured on the indicated days. n = 5 per group. Data are mean ± SEM. *p < 0.05. Data represent one of two independent experiments with similar results (n = 5 per group for each experiment). K The weight of the excised tumors was measured 25 days after cisplatin, narciclasine, or the combination. Data are mean ± SEM. *p < 0.05