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. 2017 Feb 9;8(2):e2599. doi: 10.1038/cddis.2017.8

Figure 3.

Figure 3

Combined treatment with carboplatin and thioridazine upregulated the expression of PSMA5. (a) AMC-HN4 cells were pretreated with 0.5 μM MG132 and 2.5 μM lactacystin for 30 min and were then combined with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The protein expression levels of 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 the indicated time periods. After treatment, the cells were lysed, and the proteasome activity was measured as described in the Materials and Methods section. (c) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μM thioridazine for 24 h. The protein expression levels of PSMA5 and actin were determined by western blotting. The level of actin was used as a loading control. The band intensity of the PSMA5 protein was measured using ImageJ (public domain JAVA image-processing program ImageJ (http://rsb.info.nih.gov/ij). (d) AMC-HN4 cells were transiently transfected with a control siRNA or PSMA5 siRNA. Twenty-four hours after transfection, the cells were treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, c-FLIP, Mcl-1, PSMA5, and actin were determined by western blotting. The level of actin was used as a loading control. (e) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μM thioridazine for the indicated time periods. The protein and mRNA expression levels of PSMA5 and actin were determined by western blotting and RT-PCR, respectively. (f) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were treated with 200 nM carboplatin plus 10 μM thioridazine for 24 h. The luciferase activity was analyzed. The values inb, d, and f represent the mean±S.D. from three independent samples. *P<0.01 compared with the control. #P<0.01 compared with the carboplatin plus thioridazine-treated control siRNA