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. 2016 Dec 28;11(12):e0169005. doi: 10.1371/journal.pone.0169005

Fig 4. Inhibition of IKK suppresses MβCD-induced MCP-1 expression and secretion.

Fig 4

(A) 3T3-L1 adipocytes were treated with 1 ng/ml TNFα or 4 mM MβCD for 0, 15, 30, or 60 min. (B) 3T3-L1 adipocytes were untreated (Ctrl), or treated with 1 ng/ml TNFα or 4 mM MβCD, together without or with 10 μM BMS-345541 for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. Representative immunoblots from three independent experiments are shown. (C, D) 3T3-L1 adipocytes were untreated (Ctrl), or treated with 1 ng/ml TNFα or 4 mM MβCD, together without or with 10 μM BMS-345541 for 4 h. The MCP-1 released into the media (C) and the MCP-1 protein in cell lysate (D) were determined. Each point represents the mean ± S.E. of three independent experiments. Asterisks denote significant differences (**p<0.01). (E) 3T3-L1 adipocytes were transfected with non-targeting luciferase siRNA (-) or siRNA against IKKβ (+). Protein levels of IKKβ were determined by Western blotting. (F) 3T3-L1 adipocytes were transfected with luciferase (Luc) or IKKβ siRNA. 48 h post transfection, cells were untreated (Ctrl) or treated with 4 mM MβCD for 4h. MCP-1 released into the media was determined by ELISA. Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05, **p<0.01, ***p<0.001).