Figure 3. Effects of Akt inhibition on the expression and activation of TF and TM and MT1-MMP activity in response to TNF-α.

(A and B) Effects of knockdown of Akt by siRNA on the Akt protein and mRNA levels. ECs were transfected with siRNA to Akt. The siRNA-mediated knockdown of Akt reduced Akt protein and mRNA as compared to the scrambled negative control. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. ^* P<0.05. (C–H) Effects of a specific pharmacological inhibitor of Akt on TNF-α-induced changes in TF and TM expression. ECs were treated with Akt inhibitor X (5 µmol/L) for 60 minutes or Akt siRNA and then stimulated by TNF-α for 18 hours, followed by Western blotting and TF activity assay. Western blot analyses are from an experiment representative of 4 similar experiments. TF activity was measured 6 hour after adding TNF-α (each group, n = 6). ^* P<0.05, ^** P<0.005. (I) Effects of a specific pharmacological inhibitor of Akt on on MT1-MMP activity caused by TNF-α. ECs were pretreated with Akt inhibitor X (5 µmol/L) for 60 min and then stimulated by TNF-α, and thereafter MT1-MMP activity was determined as described in the Methods section. Results are expressed as means±SD of 4 separate experiments. ^* P<0.05. The lower panel showed the protein levels of MT1-MMP as determined by Western blotting.