Figure 6.

Mitochondrial reactive oxygen species are important for carboplatin plus thioridazine-induced apoptosis. (a) AMC-HN4 cells were pretreated with 100 μM apocynin, 50 nM DPI, and 20 nM rotenone for 30 min followed by stimulation with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. (b) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μM thioridazine for 3 h (left panel) or the indicated time periods (right panel). The cells were then loaded with the Mitosox Red fluorescent dye. The Mitosox Red fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). (c) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μM thioridazine for 6 h. The cells were then loaded with the H₂DCF-DA fluorescent dye. The H₂DCF-DA fluorescence intensity was detected by flow cytometry. (d) AMC-HN4 cells were pretreated with the indicated concentrations of Mito-TEMPO and were then added with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a, b,c, and d represent the mean±S.D. from three independent samples. *P<0.01 compared with the carboplatin plus thioridazine. ^#P<0.01 compared with the control