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. 2016 Aug 25;7(40):65707–65720. doi: 10.18632/oncotarget.11604

Figure 4. Eupafolin induces up-regulation of Bim expression through AMPK-mediated inhibition of proteasome activity.

Figure 4

A. Caki cells were treated with 30 μM eupafolin for the indicated time periods. The protein and mRNA levels of Bim were determined by Western blotting, RT-PCR and real-time PCR, respectively. The level of actin was used as a loading control. B. Caki cells were treated with 30 μM eupafolin for 12 h, and then cells were washed with PBS. Caki cells treated with 20 μg/mL CHX in the presence or absence of 30 μM eupafolin for the indicated time points. The protein levels of Bim and actin were determined by Western blotting. The level of actin was used as a loading control. The band intensity of the Bim protein was measured using ImageJ (public domain JAVA image-processing program; http://rsb.info.nih.gov/ij, lower panel). C and D. Caki cells were transiently transfected with Bim siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM eupafolin and 50 ng/ml TRAIL for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry. The protein levels of PARP, Bim, and actin were determined by Western blotting. The level of actin was used as a loading control. E. Caki/ZsGreen cells were treated with 1 μM MG132 (as a positive control) and indicated concentrations of eupafolin for 24 h. Proteasome activity was analyzed by using FACS analysis. F. Caki cells were treated with 30 μM eupafolin for the indicated time periods (MG132: positive control). The protein levels of ubiquitin were determined by Western blotting. G and H. Caki cells were treated with 30 μM eupafolin for the indicated time periods. The protein levels of PSMA5, PSMB5, PSMD4/S5a, and actin were determined by Western blotting (G) and RT-PCR (H), respectively. The level of actin was used as a loading control. I. Caki cells were treated with the 30 μM eupafolin for the indicated time periods. The protein levels of phospho(p)-AMPK, AMPK, Bim, and actin were determined by Western blotting. The level of actin was used as a loading control. J. Caki cells were pretreated with the indicated concentrations with compound C for 30 min, and then added 30 μM eupaforin for 24 h. The protein levels of Bim and actin were determined by Western blotting. The level of actin was used as a loading control. K. Caki cells were transiently transfected with AMPK siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM eupafolin for 24 h. The protein levels of Bim, AMPK and actin were determined by Western blotting. The level of actin was used as a loading control. L. Caki cells were treated with 1 μM MG132 (as a positive control) and indicated concentrations of AICAR for 24 h. The cells were lysed, and the proteasome activity was measured as described in the Materials and Methods. The values in panel (C, E and L) represent the mean ± SD from three independent samples. ** p < 0.05 compared to eupafolin plus TRAIL-treated control siRNA. * p < 0.05 compared to the control.