Figure 3

Effect of endothelial protein C receptor (EPCR) blockage on the expression/activation of matrix metalloproteinases (MMPs), interleukin-1-beta (IL-1β), IL-6, and nuclear factor-kappa-B (NF-κB) by rheumatoid synovial fibroblasts (RASFs). RASFs were transfected with control (Con) or EPCR small interfering RNA (siRNA) for 24 hours. Medium was then replaced with Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS) and treated with tumor necrosis factor-alpha (TNF-α) (100 ng/mL) or activated protein C (APC) (10 μg/mL) for a further 24 hours. Media were collected for gelatin zymography to detect MMP-2 and MMP-9, and total MMP-2 and MMP-9 were semi-quantified by image analysis software in comparison with control (A) or enzyme-linked immunosorbent assay (ELISA) to quantify IL-1β (B) and IL-6 (C). Cells were collected and lysed for Western blot analysis of NF-κB or cadherin-11 (or both) in whole cell lysates (D) and in nuclei (E) and semi-quantified by image analysis software in comparison with β-actin. Std, MMP-2 and MMP-9 standard. Data on graph are shown as mean ± standard deviation (SD) of three different RASF cell lines and analyzed by using analysis of variance followed by Tukey’s honestly significant difference (HSD) post hoc test. *P <0.05, **P <0.01, compared with relevant control.