Figure 5.

TRAIL induces the phosphorylation of IκBα. (A) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cells stimulated with macrophage-conditioned medium (MaCM) were used as a positive control. The phosphorylation of IκBα was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. (B) SGBS adipocytes on day 14 of adipogenic differentiation were treated for 2 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and nuclear extracts were prepared. DNA binding activity of NFκB was analyzed by electrophoretic mobility shift assay (EMSA). One representative experiment out of three performed experiments is presented. (C) SGBS adipocytes on day 7 of adipogenic differentiation were transfected with NFκB Firefly luciferase reporter vector and Renilla luciferase control reporter vector. On day 9, cells were treated for 24 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and luciferase activity was determined. Values are means and SEM of 3 different experiments. Unpaired Student´s t-test was used to test for statistical significance. (D-H) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the IKK inhibitor SC-514 (100 μM). After 6 hours, the phosphorylation of IκBα was analyzed by Western blot (D). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, the expression of IL-6 (E), IL-8 (F), MCP-1 (G) and CCL-20 (H) was assessed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.