Data were log transformed prior to analysis by one way analysis of variance and Tukeys post hoc test, paired t tests and Students t tests, using Graphpad Software v. 4. Transfection Confluent cell cultures were detached using trypsin ethylene diamine tetraacetic acid, pelleted, and resus pended in serum free culture medium. Cells were then plated into 48 well dishes in 200l and were transfected with equal amounts of reporter plasmids. The reporter plasmids used in this study included the B reporter, comprising four tandem repeats of the B response element upstream of the firefly luciferase reporter sequence and a type II collagen enhancer luciferase reporter containing four repeats of the 48 base pair minimal enhancer of the type II col lagen gene. Each minimal enhancer sequence contains a binding site for Sox9.
Multiple repeats of the minimal enhancer are required for optimal firefly luciferase expression. Cells OC000459 ic50 were transfected with 20l serum free media containing the equivalent of 0. 156g Sox9 reporter or NFB reporter and 0. 352l Fugene 6 transfection reagent. In all experiments, chondrocytes were co transfected with a 0. 002g renilla luciferase plasmid to control for transfection efficiency. Cultures were trans fected for 4 hours prior to addition of 200l foetal bovine serum containing media. After overnight incubation, the media was aspirated off from the transfected cultures and replaced with serum free media. Cultures were treated as indicated above and collected using Passive Lysis Buffer as directed by the manufacturer.
Luciferase activity was measured using the Dual Luciferase Assay System in an L max II microplate reader. Tanscription factor regulated firefly luciferase units were adjusted relative to constitutive cytomegalovirus selleck chemical syk inhibitors regu lated renilla luciferase units obtained in control DMSO treated, U0124 treated or U0126 treated cultures. Data were log transformed prior to analysis by Students t tests and one way analysis of variance using Graphpad Software v. 4. Electrophoretic mobility shift assays Binding of nuclear protein complexes to the B or Egr 1 cog nate elements was determined as previously described. The double stranded oligodeoxynucleotides containing the B cognate sequence were purchased from Santa Cruz Biotechnology. Competition assays were performed by adding 100 fold molar excess of unlabelled probe to the nuclear extract labelled probe mixture. Antibody interference assays were performed by adding 2g antibody against Egr 1 or NFB 1 hour prior to the addition of nuclear extract to the buffered radiolabelled DNA. Samples were loaded into 4% polyacrylamide gels and were electro phoresed for 3. 5 hours. Following electrophoresis, gels were dried and exposed to Amersham Hyperfilm MP at 80 C.