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. Author manuscript; available in PMC: 2013 Jul 15.
Published in final edited form as: Clin Cancer Res. 2012 May 15;18(14):3889–3900. doi: 10.1158/1078-0432.CCR-11-3182

Figure 5. DARPP-32 abrogation of TRAIL-induced inhibition of NF-kB is associated with blocking caspases-dependent cleavage of NF-kBp65 protein.

Figure 5

A) MKN-28/DARPP-32 and MKN-28/pcDNA3 cells were treated with vehicle or 200 ng/ml TRAIL for 24h. Cells were subjected to immunofluorescence with anti-NF-kBp65 antibody and a secondary antibody conjugated to FITC. DAPI was used as a nuclear counterstain. Left panel, representative immunofluorescence images of cells following treatment with TRAIL showing localization of NF-kBp65. Cells that presented nuclear NF-kBp65 staining indicated activation of NF-kB. Right panel, the quantitative results showed that treatment with TRAIL led to a decrease of approximately 50% in nuclear NF-kBp65 in control cells relative to DARPP-32-expressing cells (p<0.0001). B) The luciferase reporter assay using a pNF-kB-Luc plasmid in MKN-28/pcDNA3, MKN-28/DARPP-32, MKN-45/control-shRNA, and MKN-45/DARPP-32-shRNA cells. After treatment with 200 ng/ml TRAIL for 24h, luciferase activity was 2.5 to 3-fold lower in MKN-28 control cells than DARPP-32-expressing MKN-28 cells (p<0.01). Knocking down endogenous DARPP-32 in MKN-45 cells and treatment with TRAIL resulted in a 3-fold decrease in luciferase activity relative to control cells (p<0.0001). C) Left panel, MKN-28/pcDNA3 and MKN-28/DARPP-32 cells were treated with vehicle or 200 ng/ml TRAIL for 24h. Western blot analysis of NF-kBp65 expression indicated that, unlike DARPP-32-expressing cells, treatment of control cells with TRAIL induced cleavage of p65 protein. Right panel, MKN-45/control shRNA and MKN-45/DARPP-32 shRNA cells were treated with vehicle or TRAIL, as in panel C. Immunoblotting data showed that knocking down endogenous DARPP-32 significantly enhanced TRAIL-induced cleavage of p65 protein as opposed to control cells. D) MKN-28 cells were treated with vehicle, 200 ng/ml TRAIL alone or in combination with 10 µM z-VAD-fmk inhibitor for 24h. Western blot results indicated that treatment with TRAIL alone induced activation of caspases and cleavage of p65. However, TRAIL in combination with z-VAD-fmk failed to activate caspases and cleave p65 protein. Down-regulation of FLIP(S) was correlated with TRAIL-induced activation of caspases and cleavage of p65. Gel loading was normalized for equal β-actin. Results are representative of three experiments and shown as the mean ± SEM. Significance of difference was calculated using Student’s t test analysis.