Fig. 2

Regulation of phosphoTwist1 nuclear localization and of Twist activity. In a and b we show representative images of Western blots, performed with nuclear, cytosol and total protein extracts of treated cells. The experiments were repeated three times with similar results. The samples were run on gel, and were processed under the same experimental conditions. B23 and vinculin were used for normalization. The numbers at the bottom indicate the fold variations for a pTwist1/Twist ratio and for b pAkt/Akt ratio versus the respective value of starved cells, considered as 1. c The cells were transiently transfected with Twist-gene reporter (TwistLuc), and co-transfected with siRNAWWOX or siRNAcontrol in the presence or the absence of WWOX-expression vector (e.v.). The histograms indicate the absolute values of Firefly/Renilla luciferase activity ratios. The data are the means ± S.E. of three independent experiments performed in triplicate. **P < 0.005, ***P < 0.001 versus TwistLuc-basal value (first bar); ^ΔP < 0.05 versus the luciferase activity in the presence of siRNAWWOX alone; ^○○P < 0.005 versus the luciferase activity value in the presence of siRNAcontrol plus WWOX e.v. Under these experimental conditions, we also show the Western blots of Wwox, Twist and pTwist1, and the experiments were repeated three times with similar results. The cytosolic and nuclear protein samples were run on gel, and were processed under the same experimental conditions. Vinculin and B23 were used for normalization. The numbers at the bottom indicate the fold variations relative to the siRNA control value, considered as 1. d The histograms indicate the fold changes of Wwox protein levels evaluated by Western blot. The normalization was performed with vinculin. The data are the means ± S.E. of three experiments. ***P < 0.001 versus basal value, considered as 1; ^○○P < 0.005 versus the value in the presence of WWOX e.v. e The cells were transiently transfected with TwistLuc, were co-transfected with WWOX e.v. or siRNAWWOX, were starved and were exposed to HGF. The histograms indicate the absolute values of Firefly/Renilla luciferase activity ratios. The data are the means ± S.E. of three independent experiments performed in triplicate. **P < 0.005, versus TwistLuc-basal value (first bar); ^ΔP < 0.05 versus the luciferase activity in the presence of HGF