Quercetin blocks bortezomib-induced Bax activation and cytochrome c release. Cells were pretreated with 20 μM quercetin and then incubated with or without 20 nM bortezomib for 12 hours. (A) Caspase activation. DoHH2 cells were used for caspase activity assay, as described in “Caspase activity assay.” *Statistically significantly increased activity of caspase as analyzed by t test (P < .001). Data are means plus or minus SD from 3 separate experiments. (B) Bax conformational change was determined in DoHH2 (left panel) and primary CLL cells (right panel) by immunoprecipitation. DoHH2 cells were treated with different concentrations of quercetin, with or without 20 nM bortezomib. The clone 6A7 anti-Bax antibody was used for the immunoprecipitation, and the clone 2D2 Bax antibody was used for Western blotting. The active Bax indicates the conformationally changed form of Bax. Western blotting for β-actin represents an equal loading of proteins. Bax translocation (C) and cytochrome release from mitochondria (D) were assessed by immunostaining in CLL cells. Mitochondria were stained with Mito-Tracker red CMRos (red). Bax was stained with the conformation-specific clone 3 Bax antibody (C), and cytochrome c was stained with cytochrome c antibody clone 6H2.B4 (D). Slides were then costained with FITC-conjugated antimouse antibody (green). Finally, the nucleus was stained with DAPI (blue). The pictures in the top panel (C) show that cells were stained with Mito-Tracker (red) and Bax, and those in the bottom panel present cells with merged DAPI staining. Arrows in panel D represent released cytochrome c in the apoptotic cells. Q indicates quercetin; B, bortezomib.