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. Author manuscript; available in PMC: 2016 May 15.
Published in final edited form as: Cancer Res. 2015 Mar 20;75(10):2049–2060. doi: 10.1158/0008-5472.CAN-14-2435

Figure 5. Z-10 and Z-12 inhibit tRXRα-mediated TNFα activation of NFκB.

Figure 5

(A) Myc-RXRα/Δ80 but not Myc-RXRα enhances TRAF2-stimulated NFκB transactivation. MCF-7 cells were transiently transfected with NFκB-Luciferase reporter and Renilla-Luciferase with or without Myc-RXRα/Δ80 and Myc-RXRα expression plasmids for 24 h. Luciferase activities were measured and normalized. The values of Y axis are 1,000 times of relative luciferase activity. (B) Myc-RXRα/Δ80 but not Myc-RXRα enhances TRAF2-induced down-regulation of IκBα. MCF-7 cells were transfected with the indicated plasmids for 24 h, and cell lysates were prepared and analyzed by immunoblotting. β-actin was used as a loading control. (C) Suppression of TRAF2 expression impairs the inhibitory effect of Z-12 on TNFα-induced IκBα degradation. MCF-7 cells transfected with siRNA of control or TRAF2 for 36 h were treated with Z-12 for 1 h before exposed to TNFα (10 ng/ml) for 30 min. Protein expressions were analyzed by immunoblotting. (D) Interaction of TRAF2 with tRXRα but not RXRα. HEK293T cells were transfected with the indicated plasmids for 24 h and then treated with TNFα (40 ng/ml) for 15 min. The complex formations were examined by co-immunoprecipitation using specific antibodies. (E) Effects of Z-10 and Z-12 on TNFα-induced formation of TRAF2/tRXRα complex. HEK293T cells were transfected with the indicated plasmids for 24 h and then treated with Z-10 (5 µM) and Z-12 (5 µM) for 1 h before exposed to TNFα (40 ng/ml) for 15 min. Protein interactions were analyzed by co-immunoprecipitation. (F) tRXRα-induced RIP1 ubiquitination is inhibited by Z-10. MCF-7 cells transfected with or without Myc-RXRα/Δ80 expression plasmids were treated with Z-10 (10 µM) for 1 h before stimulated with TNFα (20 ng/ml) for 5 min. RIP1 ubiquitination was examined by immunoprecipitated with anti-RIP1 antibody followed by immunoblotting with anti-ubiquitin antibody. Data shown are representative of at least three independent experiments. siRNA, small interference RNA; IP, immunoprecipitate.