Figure 3

16E5 expression down-regulates TGFβ1-dependent signaling. A) HaCaT E5 and HaCaT pCI-neo cells were serum starved for 12 h and then stimulated with 20 ng/ml TGFβ1 for 1 hour at 37°C. Western blot analysis shows reduction of TGFβRII protein and ligand-dependent Smad2 phosphorylation in cells expressing 16E5, while Smad2 protein levels are not affected by 16E5 expression. Densitometric analysis and Student’t test were performed as reported above *, **, ***, **** p < 0.05 vs the corresponding HaCaT pCI-neo cells. B) HaCaT E5 cells were stimulated with TGFβ1 as above. Quantitative double immunofluorescence analysis of the Smad4 nuclear translocation upon TFGβ1 treatment in E5-HA positive cells reveals that only a minor portion of the E5 positive cells show Smad4 nuclear translocation (arrowhead), while many of them display Smad4-negative nuclei (arrows). In the quantitative analysis results have been expressed as mean values ± standard errors (SE). p values were calculated using Student’s t test.* p < 0.001 vs the surrounding cells. Bar: 10 μm.