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

Figure 1.

Figure 1.

WA specifically initiates autophagy but blocks the degradation of SQSTM1 in PC cells. (A) Panc-1, SW1990, MIAPaCa-2, AsPC-1, BxPc-3 and HPDE cells were treated with increasing concentrations of WA (0.5–5 μM) for 48 h; viable cells were quantified using the MTS assay. Data are presented as mean ± SD from 3 independent experiments. (B and C) Panc-1 and MIAPaCa-2 cells were treated for 24 h with the indicated concentrations of WA, or cells were treated with 2.5 μM WA for the indicated times. Levels of protein expression were analyzed by Western blot using antibodies against LC3B and GAPDH. (D) Panc-1 cells transfected with GFP-LC3B were treated with the indicated concentrations of WA for 24 h, or treated with 2.5 μM WA for the indicated period of time. The number of GFP-LC3B dots in each cell was quantified, and at least 50 cells were included for each group. Data are presented as mean ± SD from 3 independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Scale bar: 20 μm. (E) Panc-1 and MIAPaCa-2 cells treated with DMSO (<0.1%) or WA (2.5 μM) for 24 h were imaged by transmission electron microscopy. Representative images of cells are shown. A magnified view of the electron photomicrograph shows a characteristic autophagosome. Arrowhead, autophagic vacuoles; N, nuclear. Quantification of the number of autophagic vacuoles from at least 20 randomly selected areas is shown (***, p < 0.001). Scale bar: 500 nm. (F) Panc-1 and MIAPaCa-2 cells were either untreated or treated with WA (1–2.5 μM) for 24 h in the absence or presence of Baf-A1 (100 nM), CQ (10 μM) or E64D together with pepstatin (P/E; 10 μg/ml). The indicated protein levels were analyzed by western blot. (G) Panc-1 cells were treated with DMSO (<0.1%), CQ (10 μM) or WA (2.5 μM) for 24 h followed by immunostaining with an anti-SQSTM1 antibody. Nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. (H) Panc-1 cells were pretreated with Rap (100 nM) for 30 min, followed by treatment with 2.5 μM WA or 10 μM CQ for another 24 h, and then the indicated protein levels were analyzed by Western blot.