Figure 7.

c-Jun knockdown abolishes the effect of CRBN expression on LPS-induced apoptosis upon NF-κB inhibition. A, Western blot analysis for mock- and CRBN-transfected cells with or without pretreatment with the NF-κB inhibitor BAY11-7082. THP-1 cells were differentiated by PMA (50 ng/ml) for 24 h and transfected with the pcDNA3.1 or CRBN plasmid along with siNC or sic-Jun, respectively, using Lipofectamine 2000 transfection reagent. 48 h post-transfection, cells were treated with DMSO or BAY11-7082 for 1 h prior to treatment with PBS or LPS (40 μg/ml) for 24 h. Cell lysates were immunoblotted for the indicated antibodies. B, flow cytometry analyses of mock- and CRBN-transfected THP-1 cells treated with LPS in the absence or presence of the NF-κB inhibitor BAY11-7082. Cells were treated as described in A, stained, and analyzed as described in Fig. 6B. C, quantification of apoptotic cells obtained from three biological replicates of flow cytometry analyses. Student's t test was used to calculate the p value. *, p < 0.05; ***, p < 0.001 compared with the first column (mock-transfected cells with DMSO and LPS treatment) or as indicated. #, p < 0.05; ##, p < 0.01 compared with the fifth column (pcDNA3.1- and siNC-transfected cells with BAY11-7082 and LPS treatment). ns, not significant.