Figure 2

FSTL1 activates the NF-κB pathway in FLS. (A) Following treatment, the levels of key components of NF-κB in total cell lysates were determined by western blot analysis. (B) Nuclear extracts were isolated to further confirm the activation of NF-κB pathway. In (A) and (B), FLSs were treated with FSTL1 (5 μg/ml) or vehicle (PBS solution) for 30 min. Representative western blot (Left) and quantification data (Right) are shown for the corresponding groups. The relative protein levels were normalized to the level of the internal control, actin or histone 3 (H3), and presented as fold changes relative to the control group (the level of the control group was set as 1). (C) and (D) FLSs were treated with or without FSTL1 for 12 hrs, and then ChIP assay targeting the p65 binding site on TNF-α (C) and IL-6 promoter (D) was performed. Data were shown as the percentage of input. The above results are presented as mean ± (standard deviation) SD of three independent experiments (each FLS cell line from an individual patient was used for each experiment). ^* P <0.05, ^** P <0.01, ^*** P <0.001, compared to the vehicle-treated group, Student t test. ChIP, chromatin immunoprecipitation; FLS, fibroblast-like synoviocyte; FSTL1, follistatin-like protein 1; IL-6, interleukin-6; NF-κB, nuclear factor kappa B; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor alpha.