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Figure 5


Fig. 5. Addition of active STATs does not sensitize RCS chondrocytes to FGF2-mediated growth arrest. (A) RCS chondrocytes were treated with FGF2, IL6 and IFN{gamma} for 30 minutes and analyzed for activatory phosphorylation of STAT1, STAT3 and ERK1/2 by WB. The levels of total STAT1, STAT3 and ERK1/2 serve as loading controls. (B) RCS chondrocytes were treated with FGF2, IL6 and IFN{gamma} for 72 hours and counted. The data represent an average from at least three wells with the indicated standard deviation. The arrow indicates maximal growth arrest in cells treated with 20 ng/ml of FGF2. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; results not shown on the graph) relative to untreated control, and that IL6 and/or IFN{gamma} co-treatment with FGF2 led to statistically significant differences relative to FGF2 alone (Student's t-test; *P<0.05, **P<0.01). (C) RCS chondrocytes were transfected with vector carrying constitutively activated SRC kinase (ca-SRC), grown for 24 hours and analyzed for the indicated molecules by WB. (D) RCS chondrocytes were transfected with empty vector or vector carrying ca-SRC together with STAT firefly luciferase (F-Luc) reporter vector (pTATA-TK-Luc-4xM67) and a control Renilla luciferase (R-Luc) vector (pRL-TK), grown for 96 hours and analyzed for luciferase activity as described in the Material and Methods. The data represent an average of four wells with indicated standard deviation. Statistically significant differences relative to control are indicated (Student's t-test; *P<0.05, **P<0.01). (E) RCS chondrocytes were transfected with vector carrying ca-SRC, treated with FGF2 for 72 hours and counted. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; not shown on the graph) relative to untreated controls, but overexpression of ca-SRC did not alter the FGF2-mediated growth arrest. The data represent an average from three wells with the indicated standard deviation. (F,G,H) RCS chondrocytes were transfected, analyzed for transgene expression, luciferase activity and FGF2-mediated growth arrest as described above except that vectors expressing wt or constitutively active STAT3 (ca-STAT3) were used instead of ca-SRC. Note that all FGF2 treatments led to statistically significant growth inhibition (Student's t-test; P<0.01; not shown on the graph) relative to untreated controls, but overexpression of wt or ca-STAT3 did not alter the FGF2-mediated growth arrest.