Fig. 3: SPOP negatively regulates TLR4-mediated signaling pathways.
(a) Effects of SPOP on LPS-induced transcription of TNFA, IL1B and IL6 genes. THP-1cells were transfected with either an empty control vector or a Flag-SPOP plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with LPS (100 ng/mL) for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of SPOP in the stable cell lines was examined by immunoblot analysis (Right). (b) Effects of SPOP on LPS-induced cytokines of TNFα, IL-1β and IL-6. THP-1cells were transfected with either an empty control vector or a Flag-SPOP plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with LPS (100 ng/mL) for the indicated times, and then the medium was collected for ELISA assays. (c) Effects of SPOP-RNAi on expression of SPOP and on transcription of TNFA, IL1B and IL6 genes. THP-1 cells (4 × 105) were transfected with a control or the indicated SPOP-specific siRNA (2 μg each). Forty hours later, cells were treated with LPS (100 ng/mL) for the indicated times, and then total RNA was prepared for qPCR analysis. The expression of SPOP was examined by immunoblot analysis, and the SPOP bands were quantitated using ImageJ and normalized by levels of the control protein β-actin (Right). (d) Effects of SPOP-RNAi on LPS-induced cytokines of TNFα, IL-1β and IL-6. THP-1 cells (4 × 105) were transfected with a control or the indicated SPOP-specific siRNA (2 μg each). Forty hours later, cells were treated with LPS (100 ng/mL) for the indicated times, and then the medium was collected for ELISA assays. (e) Overexpression of SPOP inhibits LPS-induced signaling. The control or Flag-SPOP stable expressed cells (4 × 105) were treated with LPS (100 ng/mL) for the indicated times, then subjected to immunoblots with the indicated antibodies. (f) Knockdown of SPOP enhances LPS-induced signaling. THP-1 cells (4 × 105) were transfected with a control or the indicated SPOP-specific siRNA (2 μg each). Forty hours later, the cells were treated with LPS (100 ng/mL) for the indicated times and then subjected to immunoblots with the indicated antibodies. Graphs show mean ± SD, n = 3. *p < 0.05; **p < 0.01.
