Fig. 6

NOXA induced by cisplatin and the combination treatment augmented MCL1 degradation. A Twenty-four hours after treatment of A549 tumor spheroids with individual or combination treatments of cisplatin and narciclasine, MCL1 mRNA levels were evaluated 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 10 μM MG-132 for 1 h, followed by treatment with 10 μM cisplatin, 0.3 μM narciclasine, or their combination for 24 and 48 h. MCL1 and NOXA levels were analyzed by western blotting. Numbers below the blot represent relative levels of the proteins, normalized to GAPDH levels. Data represent one of three independent experiments with similar results. C A549 cells transfected with siNC or siNOXA were pretreated with 5 μM cisplatin, 0.1 μM narciclasine, or their combination for 24 h before treatment with 10 μg/mL cycloheximide (CHX) for the indicated time periods. MCL1 and NOXA levels were measured by western blotting. MCL1 levels were quantified and normalized to GAPDH as shown in the right panel. *p < 0.05. Data are mean ± SD from three independent experiments. D Half-life t1/2 of MCL1 was calculated using linear regression analysis on the graph (C). E A549 cells were co-transfected with the siNC or siNOXA with or without transfection of His-tagged ubiquitin (His-Ub) for 24 h, followed by the indicated treatment for 24 h. MCL1 and NOXA levels in the whole cell lysates were measured by western blotting. MCL1 levels were quantified and normalized to GAPDH as shown in the right panel. *p < 0.05 versus same treatment group with His-Ctrl and siNC, #p < 0.05 versus same treatment group with His-Ub and siNC. Data are mean ± SD from three independent experiments