Fig. 2

NOXA was identified as the candidate gene underlying the synergistic antitumor effects of cisplatin and narciclasine. A Schematic overview of the workflow for selection and validation of candidate genes. B Heatmap showing the fold changes in the expression of potential candidate genes under different treatment conditions. C NOXA-, MAFF-, and BTG3-silenced A549 tumor spheroids were treated with individual or combination treatments of cisplatin and narciclasine for 48 h, and cell viability was assessed by measuring cellular ATP content. *p < 0.05 versus combination-treated small interfering RNA for the negative control (siNC). Data are mean ± SEM from three independent experiments in triplicate. Images were taken prior to viability assay. Scale bar: 100 μm. D Under the siRNA transfection and treatment conditions described in (C), cleaved caspase-7 (cCASP7) levels were analyzed to assess apoptosis. Data represent one of three independent experiments with similar results. E Twenty-four hours after treatment of tumor spheroids with individual or combination treatment of cisplatin and narciclasine, the mRNA levels of NOXA, MAFF, and BTG3 were assessed using RT-qPCR. Data are presented as fold change in gene expression, normalized to GAPDH expression. *p < 0.05 versus vehicle. Data are mean ± SEM from three independent experiments in triplicate. F Following 48 h of treatment under the indicated treatment conditions, protein levels of NOXA, MAFF, and BTG3 were assessed using western blotting. β-Actin and GAPDH were used as the loading control. Experiments were conducted in triplicate. Data represent one of three independent experiments with similar results