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. 2017 Feb 9;31(5):2013–2025. doi: 10.1096/fj.201600976R

Figure 5.

Figure 5.

In colon cells, TNF-α-stimulated NF-κB depends on reduced mitochondrial function leading to AMPK activation. A) Luciferase activity (relative luciferase unit, RLU) driven by NF-κB promoter in HCT116 control (con) cells transfected with pNFKB-MetLuc2-reporter vector with and without TNF-α (4 h) treatment in presence of AntiA and CompC (n = 3). *P < 0.05, compared to con; #P < 0.05 compared to TNF-α alone, ANOVA. B) Colonic ρ0 cells were transfected with pNFKB-MetLuc2-reporter vector and treated with TNF-α (4 h) with and without CompC; relative luciferase activity (RLU) (n = 3). *P < 0.05, compared to con, Student’s t test. C) HCT116 con cells were transfected with pNFKB-MetLuc2-reporter vector and siRNA for AMPKα2; TNF-induced (4 h) relative luciferase activity (RLU) (n = 3). Total protein of negative con (neg) and AMPKα2 siRNA transfected cells was immunoblotted for AMPKα2. *P < 0.05 compared to con, Student’s t test. D) In HCT116 con cells in presence of AntiA and ρ0 cells, IκBα protein levels were assessed by immunoblotting (densitometric analysis of n = 3). *P < 0.05; *P < 0.05 compared to con, ANOVA. E) In HCT116 con cells transfected with negative con (neg) and AMPKα2 siRNA, total protein was analyzed for IκBα and AMPKα2 (densitometric analysis of n = 3). *P < 0.05, compared to con, Student’s t test. F) Nuclear protein fraction of HCT116 con and ρ0 cells stimulated with TNF-α (15 min) were immunoblotted for phosphorylated p65 (p-p65), total p65, and poly(ADP-ribose)polymerase (PARP) as loading con (densitometric analysis of n = 3). *P < 0.05, compared to con, #P < 0.05 compared to TNF-α alone, ANOVA. G) Total protein of HCT116 ρ0 cells treated with TNF-α (15 min) in presence of CompC were immunoblotted for phosphorylated p65 (p-p65) and total p65 (densitometric analysis of n = 3). *P < 0.05, compared to con; #P < 0.05 compared to TNF-α alone, ANOVA. H) HCT116 con cells transfected with negative control (neg) and AMPKα2 siRNA, were treated with TNF-α (15 min) and total protein was immunoblotted for phosphorylated p65 (p-p65) and total p65 and (densitometric analysis of n = 3). *P < 0.05, compared to con; #P < 0.05 compared to TNF-α alone, ANOVA.