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. 2007 Aug;171(2):513–524. doi: 10.2353/ajpath.2007.070188

Figure 6.

Figure 6

Suppression of autophagy enhances proteasome inhibitor-induced ER stress in cancer cells. A: Bax-positive (a–c, e–g) and Bax-deficient (d and h) HCT 116 cells were treated with MG132 (1 μmol/L) in the presence or absence of z-VAD (50 μmol/L) as indicated and analyzed 24 hours later by phase microscopy (a–d) and Hoechst staining (e–h). Arrows indicate the apoptotic cells (f), which were greatly reduced in the presence of z-VAD (g) or in the absence of Bax (h). Note that cellular vacuolization was not affected by z-VAD or Bax. B: Bax-deficient HCT 116 or DU145 cells were transfected with siRNA against the indicated Atg genes or a negative control siRNA (Neg) for 48 hours before they were treated with MG132 (0.25 or 0.5 μmol/L, respectively). Percentage of vacuolated cells was quantified 24 hours later. * and #P < 0.01; $P < 0.05 by Z-test (MG132 plus specific siRNA versus MG132 plus negative siRNA in each cell line. * and $HCT 116 cells; #DU145 cells). C: HCT 116 Bax-negative cells were treated with lactacystin (5 μmol/L), ALLN (10 μmol/L), or bortezomib (20 nmol/L) for 24 hours in the presence (closed column) or absence (open column) of 3-MA (10 mmol/L). Percentage of cells with vacuoles was determined. *P < 0.01 by Z-test (groups without 3-MA versus groups with 3-MA for each proteasome inhibitor). D: Bax-deficient HCT 116 cells were treated with vehicle control (a) or MG132 (1 μmol/L b) for 24 hours followed by immunostaining with an anti-calnexin antibody. E: DU145 (a and b) or Bax-deficient HCT 116 cells (c and d) were treated with MG132 (1 or 5 μmol/L, respectively) for 24 hours and then analyzed by electron microscopy. Representative images are shown to indicate the progressive dilation of the ER lumen. The boxed area in a is enlarged in b. Arrows indicate the dilated segment of selected ER (b). Scale bars: 0.25 μm (a and b), 2 μm (c and d). N, nucleus. F: HCT 116 cells were treated with MG132 (1 μmol/L) for indicated times followed by immunoblot analysis. G: Bax-deficient HCT 116 (a and b) and DU145 (c and d) cells were transfected with siRNA against Atg8/LC3B (b and d) or a negative siRNA (a and c) for 36 hours before they were treated with MG132 (0.1 or 0.25 μmol/L, respectively) for 16 hours. Representative electron micrographs are shown to indicate the suppression of autophagy and the enhancement of ER dilation in Atg8/LC3B-knocked-down cells (b and d). N, nucleus; black arrows, autophagic vacuoles; white arrows, nondilated ER. Scale bar = 1 μm. H: Bax-deficient HCT 116 cells were transfected with the indicated siRNA and treated with MG132 as in F. Total cell lysates were prepared, and caspase-4 activity was measured using Ac-LEVD-AFC as the substrate. The results were expressed as the fold of increase over the nontreated control group.