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. 2013 Oct 11;288(47):33654–33666. doi: 10.1074/jbc.M113.518134

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — Modulation of p62/sequestosome 1 expression sensitizes colon cancer cell lines to undergo apoptosis by ABT-263. A, HCT116 cells were stably transfected with a lentiviral GFP-LC3B to fluorescently label autophagosomes. Autophagy induction is indicated by an increase in the number of LC3B puncta compared with a diffuse staining pattern in vehicle-treated (dimethyl sulfoxide, DMSO) cells (top). To confirm autophagy induction, autophagic flux was studied using bafilomycin (baf) A1 with analysis of the conversion of LC3I to its lipidated form, LC3II (bottom). Densitometry was performed to quantify protein bands and normalized to tubulin. B, effect of chloroquine (CQ, 25 μm; 48 h) pretreatment on caspase cleavage (CL) induced by ABT-263 (24 h) in whole cell lysates from HCT116 and DLD1 cell lines. Tubulin serves as control for protein loading. C, effect of autophagy inhibition by bortezomib (bort) alone (12) and combined with chloroquine on p62 expression. D, effect of bortezomib on ABT-263-induced (24 h) caspase cleavage in the presence of chloroquine. E, cells containing stable expression of p62 shRNA or control were treated with ABT-263 (24 h) alone or combined with chloroquine and/or bortezomib. LC3 expression and caspase cleavage products were analyzed by immunoblotting. *, nonspecific band.