Figure 2
Blocking endothelial protein C receptor (EPCR) suppresses rheumatoid synovial fibroblast (RASF) survival/growth, migration/invasion, and the ability to degrade cartilage. (A) EPCR mRNA levels in RASFs transfected with EPCR or control small interfering RNA (siRNA) for 24 hours and detected by reverse-transcription real time polymerase chain reaction (PCR). (B) EPCR protein in whole cell lysates of RASFs transfected with EPCR or control siRNA for 72 hours and detected by enzyme-linked immunosorbent assay (ELISA). (C) Viability of RASFs transfected with control or EPCR siRNA or EPCR blocking (RCR252) or non-blocking (RCR92) antibody for 72 hours and detected by colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue exclusion assays. (D) Migration/invasion of RASFs transfected with control or EPCR siRNA for 72 hours. After siRNA transfection for 24 hours, RASFs were embedded into collagen gels at 1 × 105 cells/mL. After incubation for another 48 hours, collagen gels were stained and cells that had migrated outside of gels were counted (16 gel drops for each treatment). (E) Cartilage degradation by RASFs transfected with control or EPCR siRNA. After 24 hours of siRNA transfection, OA cartilage was co-incubated with RASFs for another 24 hours, and culture medium was then collected for measuring sulphated glycosaminoglycans (sGAGs) by a 1,9-dimethylmethylene blue (DMMB) assay. Cartilage explants were fixed and processed for toluidine blue staining. Data on each graph are shown as mean ± standard deviation (SD) from four RASF cell lines derived from four RA synovial tissues and analyzed by one-way analysis of variance followed by Tukey’s honestly significant difference (HSD) post hoc test (C) or Student t test (A,B,D,E), compared with control siRNA. The images represent one of three experiments Scale bar: 100 μm. *P <0.05, **P <0.01.