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. 2018 Sep 13;20(10):1045–1058. doi: 10.1016/j.neo.2018.08.008

Figure 6.

Figure 6

PDZ-RhoGEF (PRG) enhances TROY-induced NF-κB activation. (A) 293 cells expressing a NF-κB-luciferase reporter (293/NF-κB-luc) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were serum starved (0.1% BSA) for 16 hours and lysed, and NF-κB-luc reporter expression was measured using a luciferase reporter assay kit. Luciferase activity was normalized to the vector-transfected cells. The data are depicted as the mean values (+/− SD) (n = 3, *P < .05; **P < .01) (upper panel). The expression of HA-tagged TROY and Myc-tagged PDZ-RhoGEF in lysates was detected by immunoblotting (bottom panel). (B) 293/NF-κB-luc reporter cells overexpressing TROY (293/NF-κB-luc/TROY-HA) were transfected with either vector or 0.8 or 1.6 μg Myc-tagged PDZ-RhoGEF. Twenty-four hours after transfection, cells were serum starved (0.1% BSA) for 16 hours and lysed, and luciferase activity was measured using luciferase reporter assay kit. Luciferase activity was normalized to the vector-transfected cells. The data are depicted as the mean values (+/− SD) (n = 3, ***P < .001) (upper panel). Expression levels of PDZ-RhoGEF and TROY were determined by immunoblot analysis (bottom panel). (C) 293/NF-κB-luc/TROY-HA cells were transfected with either two independent siRNAs targeting RhoC or RhoA or a control siRNA (siCTRL). Twenty-four hours after transfection, cells were transfected with Myc-tagged PRG plasmid for an additional 24 hours. Cells were serum starved (0.1% BSA) for 16 hours and lysed, and luciferase activity was measured using a luciferase reporter assay kit. Luciferase activity was normalized to the control cells. The data are depicted as the mean values (+/− SD) (n = 3, *P < .05; **P < .01; ***P < .001) (upper panel). Knockdown of RhoC and RhoA expression and overexpression of PDZ-RhoGEF was verified by immunoblotting (bottom panel). (D) T98G/NF-κB-luc/TROY-HA cells were transfected with either two independent siRNAs targeting RhoC or RhoA or a control siRNA (siCTRL). Twenty-four hours after transfection, cells were serum starved (0.1% BSA) for additional 16 hours and lysed, and luciferase activity was measured using a luciferase reporter assay kit. Luciferase activity was normalized to the control cells. The data are depicted as the mean values (+/− SD) (n = 3, **P < .01; ***P < .001) (upper panel). Knockdown of RhoC and RhoA expression was verified by immunoblotting (bottom panel).