Figure 3.

Role of PI3K in the ACPA-induce synovial fibroblast migration. Starved FLS were exposed to 1 µg/mL polyclonal ACPA IgG (ACPA) or non-ACPA control IgG (IgG) or to TNF 10 ng/mL. Immunoblot analysis of Akt (Thr308 or Ser473 residues), ERK1/2, JNK and p38 phosphorylation are shown, whereas GAPDH protein was detected as control. The presented data are representative for at least three independent experiments for all tested proteins (A). Change of Akt phosphorylation at the Thr308 residue is shown in response to 1 µg/mL ACPA or IgG treatments (B). The data were calculated following densitometry analysis in five independent experiments, levels were normalised to baseline intensities, mean±SD values are shown. FLS were pretreated with 200 nM of the PI3K inhibitor wortmannin (C) or 2 µg/mL of the PTEN inhibitor SF-1670 (D) for 2 hours prior to the analysis of cell migration in the presence of 1 µg/mL ACPA or control IgG. DMSO (0.01%) was used as solvent control. Graphs represent fold-change in cell mobility, mean±SD values were calculated from three independent experiments, *P<0.05. ACPA, anticitrullinated protein/peptide antibody; DMSO, dimethyl sulfoxide; FLS, fibroblast-like synoviocytes; PAD, protein arginine deiminase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; ns, not significant; TNF, tumour necrosis factor.