Fig. 2.

Curcumin enhances PP242-induced apoptosis. a Caki cells were treated with 20 μM of curcumin, kahweol, quercetin, luteolin, and triptolide in the absence or presence of 0.5 μM PP242 for 30 h. b–d Caki cells were treated with 20 μM curcumin in the absence or presence of 0.5 μM PP242 for 30 h. The nuclei were detected by DAPI staining. Scale bar = 20 μm (b). The cytoplasmic histone-associated DNA fragments were measured (c). Caspase activities were calculated using caspase-3 (DEVDase) assay kits (d). e Caki cells were treated with 20 μM curcumin plus 0.5 μM PP242 for 30 h in the presence or absence of 20 μM z-VAD-fmk (z-VAD). Immunoblot analysis of PARP and actin. f–j Caki cells were treated with 20 μM curcumin plus 0.5 μM PP242 for the indicated time periods. The mitochondrial membrane potential was measured using rhodamine123 fluorescent dye by flow cytometer (f). Cytosolic fractions were analyzed for cytochrome c release. The level of MnSOD was used as a mitochondria loading control (g). Cells stained for active Bax using conformation-specific antibodies. Flow cytometry (h) and fluorescence microscopy (i) detect fluorescence intensity. Scale bar = 20 μm (i). For Bax oligomerization assay, monomers and oligomers of Bax were quantified by western blotting (j). k Caki cells were treated with 20 μM curcumin in the presence or absence of 0.5 μM PP242 for 30 h. Immunoblot analysis of cIAP2, DR5, c-FLIP, Mcl-1, Bcl-2, Bcl-xL, Survivin, Bim, and actin. l Vector-transfected cells (Caki/Vec) and Mcl-1-(Caki/Mcl-1) or Bcl-2-overexpressing cells (Caki/Bcl-2) were treated with 20 μM curcumin in the presence or absence of 0.5 μM PP242 for 30 h. Immunoblot analysis of PARP, Mcl-1, Bcl-2, and/or actin. The data represented in a, c, d, e, f, and l are the means ± s.d. from three independent samples. **p < 0.01 compared to the control. #p < 0.01 compared to the combined treatment with PP242 plus curcumin. *p < 0.01 compared to the combined treatment with PP242 plus curcumin-treated Caki/Vec