Figure 4

Focal adhesion kinase depletion reduces murine arthritic synovial fibroblast invasion, but not migration, in vitro . TNF⁺CreER⁺FAK^f/f murine synovial fibroblasts were treated with either (Z)-4-OH tamoxifen (OHT) or 95% ethanol vehicle control (EtOH). Cell lysates were subjected to Western blot analysis for focal adhesion kinase (FAK) and actin to test for FAK depletion. (A) A representative blot from seven experiments is presented. (B) Densitometry was performed, and the average ± SEM for data for the amount of FAK divided by actin are shown (n = 7, ****P < 0.0001 by t-test). (C) TNF⁺CreER⁺FAK^f/f (+CreER) and TNF⁺CreER^-FAK^f/f or TNF⁺CreER^-FAK^+/+ (-CreER) murine synovial fibroblasts were treated with OHT or EtOH for 4 days and then allowed to invade for 24 hours through Matrigel invasion chambers. The number of invaded cells per microscopic field at 100× magnification was counted, and OHT-treated samples were normalized to EtOH-treated controls. Graph depicts average ± SEM (n = 3, **P < 0.01, ***P < 0.005 by one-way analysis of variance (ANOVA)). (D) +CreER and -CreER murine synovial fibroblasts were treated as in (C) and allowed to migrate across fibronectin-coated transwells for 4 hours. The number of migrated cells per microscopic field at 100× magnification was counted, and OHT-treated samples were normalized to EtOH-treated controls. Graph depicts average ± SEM data (n = 3). (E) Murine TNFα-overexpressing synovial fibroblasts were treated with dimethyl sulfoxide (DMSO) vehicle control, PF-562,271 or PF-573,228 and allowed to migrate across fibronectin-coated transwells for 4 hours. The number of invaded cells per microscopic field at 100× magnification was counted. Graph depicts average ± SEM data (n = 4, *P < 0.05 by one-way ANOVA).