Fig. 5. Effects of pretreatment with N-acetylcysteine on apigenin-induced cytotoxicity in 3D cultures of MSTO-211H and H-2452 cells. Spheroids were cultured in ultralow cluster 96-well plate and pretreated with or without 5 mM NAC 2 h prior to treatment with 30 μM apigenin for 48 h. (A) Vitality staining of spheroids (from left to right: phase-contrast image [a], fluorescent images of FDA(+) living cells in green [b], PI(+) dead cells in red [c], and merged [d]) (×100). (B) Cell and spheroid viability were measured by MTT assay and the enhanced cell viability assay kit. (C) The levels of necroptosis- and apoptosis-related proteins were analyzed by Western blotting. APG, apigenin; NAC, N-acetylcysteine. *p < 0.05 vs. respective control group. #p < 0.05 vs. group treated with APG alone.
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