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

Figure 5. Effects of C/EBPβ siRNA on the Saa3 promoter activity in response to activated RAW264.7 macrophages.

Figure 5

(A) 3T3-L1 preadipocytes were treated with MDI for 48 h and differentiated into mature adipocytes as described under “EXPERIMENTAL PROCEDURES”. Differentiated 3T3-L1 cells were transfected with control siRNA (si-cont) or C/EBPβ siRNA (si-C/EBPβ). After 2 days of transfection, total RNAs were extracted and subjected to quantitative PCR analysis (A) to examine the expression level of C/EBPβ mRNA. The level of L19 transcript was used as a control. (B,C) After 2 days of transfection, differentiated 3T3-L1 cells were subjected to the co-culture system with RAW264.7 cells. Saa3 mRNA level (B) and luciferase activity (C) in the 3T3-L1 adipocytes was examined upon co-culture with macrophages activated by LPS. *p < 0.05. (D) After 2 days of transfection, 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) After 2 days of transfection, 3T3-L1 adipocytes were treated with 10 ng/ml of TNF-α for 24 h. Luciferase activity in the 3T3-L1 adipocytes was examined. (F) Mature 3T3-L1 cells were treated with MacCM for 5,10,15 minutes. p-CEBP/β, CEBP/β and β-actin proteins were detected by western blot analysis. The data (mean ± S.E.) are from a single experiment carried out (n = 3) and are representative of two independent experiments. *p < 0.05.