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. 2016 Jun 16;12(9):1521–1537. doi: 10.1080/15548627.2016.1191722

Figure 4.

Figure 4.

WA inhibits proteasome activity and induces ER stress-related apoptosis in PC cells. (A and B) WA affected ubiquitin-proteasomal activity in PC cells. Panc-1 and MIAPaCa-2 cells were treated with either DMSO (<0.1%) or the indicated concentrations of WA for 24 h, followed by measuring inhibition of the proteasomal chymotrypsin (CT)-like activity using a cell-based assay (A) and Western blot analysis using specific antibodies to ubiquitin (B). Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (C) Panc-1 and MIAPaCa-2 cells were treated with DMSO (<0.1%) or WA (2.5 μM) for 24 h followed by immunostaining with an anti-CANX antibody. Nuclei are counterstained with DAPI (blue). Arrows indicate dilated ER cavities. Scale bar: 10 μm. (D) Western blot analysis of ER stress-related protein levels after Panc-1 and MIAPaCa-2 cells were treated with the indicated concentrations of WA or tunicamycin (TM, 10 μg/ml) for 24 h. (E) Apoptotic cells were detected by flow cytometry using an ANXA5-FITC staining assay after cells were treated as described in (A). Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (F) Western blot analysis of PARP1, CASP9, CASP8 and cleaved (C)-CASP3 levels after cells were treated as described in (A). (G) Panc-1 cells were pretreated or not with CHX (1 mg/ml) for 1 h, followed by treatment with the indicated concentrations of WA for 24 h. The ubiquitinated protein levels (upper panel) and the cell viability (lower panel) were determined by Western blot and MTS assay, respectively. Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05). (H) Panc-1 and MIAPaCa-2 cells were pretreated with TUDCA (1 mM) for 30 min, and then exposed to WA (2.5 μM) for 24 h. The indicated protein levels (upper panel) and the apoptotic cells (lower panel) were determined by Western blot and ANXA5-FITC staining assay, respectively. Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05). (I) Panc-1 cells were transfected with DDIT3 siRNAs (#1 and #2) for 48 h and then cells were treated with WA (2.5 μM) for an additional 24 h. The indicated protein levels (upper panel) and the apoptotic cells (lower panel) were determined by Western blot and ANXA5-FITC staining assay, respectively. Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05).