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. 2015 Dec 17;18(6):840–848. doi: 10.1093/neuonc/nov299

Fig. 3.

Fig. 3.

Marizomib increases reactive oxygen species (ROS) generation, but N-acetyl cysteine (NAC) quenches ROS induction, blocks caspase-3 activation, and improves the survival of D-54 cells. (A) After treatment with marizomib (60 nM) for 12 hours, ROS generation was measured as the number of DCF fluorescent cells by direct fluorescent microscopy. The percentage of ROS-positive cells is shown in the lower panel. (B) Carboxy-H2DCFDA detectable ROS were measured by fluorescence spectroscopy. Values represent the mean ± SD of 3 experiments. (C) The addition of N-acetyl cysteine (NAC) (10 mM) blocked the marizomib-induced ROS activation. (D) NAC pretreatment was able to rescue the D-54 cells from marizomib-induced cell death. (2-way ANOVA analysis, P < .0001). (E) The caspase-3 activation triggered by marizomib was abolished by NAC pretreatment (2-way ANOVA analysis, P < .0001). (F) D-54 cells were treated with 20 or 60 nM marizomib in the presence or absence of 10 mM NAC for indicated time points. Western blot was used to detect cleaved caspase-3 and PARP. Actin was used as the internal control. **P < .01, ***P < .001, ns = not significant.