Figure 3

cAMP suppresses TNF-α-induced cell death in L929 cells via CREB-mediated transcription. A, L929 cells were treated with forskolin (10 μM), PGE₂ (10 μM), epinephrine (100 μM), or 6-MB-cAMP (100 μM) for 30 min. Phosphorylation of CREB and expression of CREB and actin were analyzed by immunoblotting (IB). B, L929 cells were treated with or without actinomycin D (ActD, 1 μg/ml, 30 min) prior to forskolin treatment (10 μM, 30 min), followed by stimulation with or without 2 ng/ml TNF-α for 12 h. Cell death was measured by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments. C, L929 cells were transfected with CREB siRNAs or the negative control (NC) siRNA (200 nM each). After 72 h, cells were stimulated with or without forskolin (10 μM, 30 min). Phosphorylation of CREB and expression of CREB and actin were measured by immunoblotting. D, L929 cells were transfected with CREB siRNAs or the negative control siRNA (200 nM each). After 48 h, cells were pretreated with or without forskolin (10 μM, 30 min), followed by stimulation with or without 10 ng/ml TNF-α for 24 h. Cell death was monitored by PI staining. The percentages of cell death are shown in the right panel as mean ± SD; n = 3. Left panel is representative of three independent experiments.