Fig 2: Combined treatment of IL-1ß and TNFa increases CREB binding at IL-6 promoter. (A) IL-6 promoter contains an adjacent CREB and C/EBPß binding sites. Chromatin from adipocytes treated with IL-1ß, TNFa alone or in combination was subjected to ChIP with antibodies against (B) CREB or (C) C/EBPß followed by qRT-PCR. CREB or C/EBPß occupancy at IL-6 promoter was determined. Data are expressed as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01.

Fig 3: Schematic illustration of signaling pathway underlying IL-1β/TNFα-induced expression of IL-6 in adipocytes.

Fig 4: Combined effect of IL-1ß and TNFa on IL-6 expression in mouse adipocytes. (A,B) 3T3 L preadipocytes were differentiated into adipocytes as described in materials & methods. Lipid droplets in adipocytes were determined by using Nile Red staining. Morphology of adipocytes and adipogenic markers were shown. Scale Bar 50 µm. Mouse 3T3-L adipocytes were stimulated with IL-1ß (10 ng/mL) and TNFa (10 ng/mL) alone or in combination for 24 h. Cells and culture media were collected. (C) Total RNA was extracted from the cells and IL-6 mRNA was quantified by real time PCR. Relative mRNA expression was expressed as a fold change. (D) Secreted IL-6 protein in culture media was determined by ELISA. (E) Different number of cells (1, 0.5, 0.25 million) were treated with IL-1ß (10 ng/mL) and TNFa (10 ng/mL) alone or in combination for 24 h. Cells and culture media were collected, Secreted IL-6 protein in culture media was determined by ELISA. (F) 3T3 adipocyte cells were stained for confocal microscopy, as described in the Materials and Methods section. IL-6 expression is shown by green fluorescence (inset), whereas nuclei are stained blue with DAPI (original magnification ×40). Scale Bar 20 µm. (G) IL-6 fluorescence intensity was determined for 10 random images. The results obtained from three independent experiments are shown. All data are expressed as mean ± SEM (n = 3). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 5: Trichostatin A (TSA) further enhances IL-1ß/TNFa expression of IL-6. Cells were treated with TSA (10 nM) for 4 h before stimulation with vehicle, IL-1ß, TNFa, or IL-1ß + TNFa for 24 h. (A,B) IL-6 mRNA and secreted protein were determined by qRT-PCR and ELISA, respectively. Data were expressed as mean ± SEM. **** p < 0.0001.