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. 2016 Dec 8;6:38697. doi: 10.1038/srep38697

Figure 4. C/EBPβ is involved in Saa3 promoter activity in adipocytes in response to activated RAW264.7 macrophages.

Figure 4

(A) HEK293T cells were transiently transfected with constructs for Saa3-luc with pcDNA-C/EBPβ or empty vector. After 2 days of the transfection, cells were treated with 10 ng/ml of TNF-α for 8 h, scraped and subjected to luciferase as described in Experimental Procedures. Data are means of triplicate experiments (mean ± S.E.). (B) Putative three C/EBPβ binding elements are located in the mouse Saa3 promoter region (−314/+50). Saa3 promoter was mutated and ligated upstream of the complete luciferase cDNA, generating mutant Saa3 promoter-luciferase chimeric genes (mut1, mut2 and mut3), respectively. (C) 3T3-L1 adipocytes were infected with retroviruses harboring each mutant promoter gene. A co-culture system composed of infected adipocytes and RAW264.7 macrophages were constructed. Luciferase activity in the 3T3-L1 adipocytes was examined upon co-culture with macrophages activated by LPS. *p < 0.05. (D) Infected 3T3-L1 adipocytes were treated with conditioned medium of RAW264.7 cells without LPS (none) or stimulated in the presence of 1 μg/ml of LPS for 18 hr (LPS). (E) Infected 3T3-L1 adipocytes were treated with 10 ng/ml of TNF-α for 24 h. Luciferase activity in the 3T3-L1 adipocytes was examined. The data (mean ± S.E.) are from a single experiment carried out (n = 3) and are representative of two independent experiments. *p < 0.05, ** p < 0.01.