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. 2005 Jun 27;102(27):9631–9636. doi: 10.1073/pnas.0504097102

Fig. 3.

Fig. 3.

Effects of cardiac glycosides on TNF-induced IKK and JNK activation and the function of upstream or downstream kinases. (a) HeLa cells were pretreated with 100 nM oleandrin (I), 100 nM digitoxin (II), or 2,000 nM inactive cardiac glycoside (VIII) for 6 h. Cells were then treated with 30 ng/ml TNF-α for 5 min. Cell extracts were used for the IKK kinase and Western blot assays. E, 0.1% ethanol control; 0, no drug or carrier additions. (b) Reporter plasmids pNF-κB-luc and pRL-CMV were cotransfected into HeLa cells with effector plasmids HA-IKKα, HA-IKKβ, HA-p65 overnight, then treated with 100 nM oleandrin (I) for 10 h. Cell extracts were tested with the dual-luciferase reporter assay system. (c) Reporter plasmids pNF-κB-luc and pRL-CMV were cotransfected into HeLa cells with effector plasmids HA-MEKK3 and Flag-NIK overnight, then treated with 100 nM oleandrin (I) for 10 h. Cell extracts were then tested with the dual-luciferase reporter assay system. (d) HeLa cells were pretreated with 100 nM oleandrin (I), 100 nM digitoxin (II), or 2,000 nM inactive cardiac glycoside (VIII) for 6 h, then treated with 30 ng/ml TNF-α for 15 min. Cell extracts were tested by using the JNK kinase and Western blot assays.