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. 2010 May 5;151(7):3407–3419. doi: 10.1210/en.2009-1321

Figure 6.

Figure 6

Effects of dominant-negative C/EBP, C/EBPβ siRNA, and dominant-negative CREB on cAMP-, TNF-, and p65-induced SAA3 promoter activity. Panel A, Effect of dominant-negative C/EBP on TNF-induced SAA3 promoter activity in mouse granulosa cells. DN, Dominant-negative. Panel B, Effect of dominant-negative C/EBP on interaction between cAMP and p65 in increasing SAA3 promoter activity. Cells were transfected for 3.5 h with mSAA3-172LUC alone or with dominant-negative C/EBP vector (100 ng/ml) and p65 expression vector (10 ng/ml) and then incubated with TNF (10 ng/ml) alone and/or with cAMP (500 μm) for 16 h. The luciferase activity was normalized using total protein concentrations and expressed as a fold change in comparison with the control. DN, Dominant negative; LUC, luciferase. Panel C, Effects of TNF and cAMP on C/EBP isoforms in mouse granulosa cells. Cells were cultured with TNF (10 ng/ml) and cAMP (500 μm) alone and in combination for 24 h. Each lane was loaded with 20 μg of total protein using whole-cell lysates. β-Actin was used as a loading control. C, Control; A, cAMP; T, TNF; TA, TNF+cAMP. Panel D, Inhibitory effect of C/EBPβ siRNA on cAMP and TNF in enhancing SAA3 promoter activity. Cells were transfected for 24 h with C/EBPβ siRNA (10 nm) and then further transfected for 3.5 h with mSAA3-172LUC. Transfected cells were incubated with TNF (10 ng/ml) alone, cAMP (500 μm) alone, and in combination for 16 h. Panel E, Effect of C/EBPβ siRNA on C/EBPβ, SAA3, phospho-IκB, IκB, and p65 protein expression after cAMP and TNF treatments in mouse granulosa cells. Cells were transfected for 24 h with C/EBPβ siRNA (10 nm) and then incubated with TNF (10 ng/ml) and cAMP (500 μm) in combination for 24 h. Panel F, Effect of cAMP and TNF on pCRE-luc promoter activity in granulosa cells. Cells were transfected for 3.5 h with pCRE-luc and then incubated with TNF (10 ng/ml) alone, cAMP (500 μm) alone, and in combination for 16 h. Luciferase activity was normalized using total protein concentrations and expressed as a fold change in comparison with the control. Panel G, Effect of dominant-negative CREB on interaction between cAMP and TNF in increasing SAA3 promoter activity. Cells were transfected for 3.5 h with mSAA3–172LUC alone or with dominant-negative CREB vector (100 ng/ml) and then incubated with TNF (10 ng/ml) alone and/or with cAMP (500 μm) for 16 h. Luciferase activity was normalized using total protein concentrations and expressed as a fold change in comparison with the control. Different color bars indicate significant differences (P ≤ 0.05) among groups. DN, Dominant-negative; LUC, luciferase.