Fig. 4

Narciclasine and the combination treatment reduced MCL1 levels through inhibition of translation. A A549 tumor spheroids were treated with 0.3 μM narciclasine for the indicated time periods, and MCL1 mRNA levels were quantified using RT-qPCR. Data are presented as fold change in MCL1 expression, normalized to GAPDH expression. *p < 0.05. Data are mean ± SEM from three independent experiments in triplicate. B A549 tumor spheroids were pretreated with 0.3 μM narciclasine for 2 h before treatment with 10 μM MG-132 for the indicated time periods. MCL1 protein levels were analyzed by western blotting. Data represent one of three independent experiments with similar results. C A549 tumor spheroids were treated with 10 μg/mL cycloheximide and 0.3 μM narciclasine for the indicated time periods. Data are mean ± SD from three independent experiments. D Protein synthesis was assessed using surface sensing of translation assay. A549 tumor spheroids were treated as indicated doses of narciclasine for 2 h, followed by incubation with 5 μg/mL puromycin for 15 min. For the right panel, cisplatin and narciclasine were treated at concentrations of 10 and 0.3 μM, respectively. Whole cell lysates were obtained and subjected to western blotting using anti-puromycin antibody. Data represent one of three independent experiments with similar results. E A549 tumor spheroids were treated with 0.3 μM narciclasine for the indicated time periods. Levels of phospho-eIF2α, phospho-4E-BP1, and other proteins involved in the unfolded protein response (UPR) pathway were analyzed by western blotting. Data represent one of three independent experiments with similar results. F Whole cell lysates were prepared from A549 tumor spheroids treated with 10 μM cisplatin, 0.3 μM narciclasine, or the combination for the indicated time. Levels of proteins involved in the UPR pathway were analyzed by western blotting. Data represent one of three independent experiments with similar results