Fig. 4. Effect TGFβ on the gene expression under control of p15ink4b promoter.

A. MEE cells were transiently transfected with pGL3-p15-Lux wild type plasmid and/or plasmids containing mutations in the Smad-binding sites. After transfection, the cells were incubated with TGFβ1 (2 or 5ng/ml) for 24 hours. Background luciferase activity (empty vector) was subtracted from all data. Error bars indicate standard deviation from at least three independent experiments. TGFβ1 increases p15ink4b promoter activity by threefold in MEE cells. TGFβ1 effect is Smad4-dependent, since addition of TGFβ1 to MEE cells transfected with plasmids containing mutations in Smad binding sites did not increase luciferase activity. All three SBEs in p15ink4b promoter are implicated in TGFβ1 induced promoter activation

B. MEE cells were transiently transfected as describe in figure legend 3A. After transfection, the cells were incubated with TGFβ3 (2 or 5ng/ml) for 24 hours. Background luciferase activity (empty vector) was subtracted from all data. Error bars indicate standard deviation from at least three independent experiments. Addition of TGFβ3 does not significantly increase luciferase activity in MEE cells transfected with pGL3-p15wt-Lux plasmid as well as with the plasmids containing mutations in Smad-binding sites.

C. Endogenous TGFβ1 and TGFβ3 were blocked using the antibody (anti TGFβ1 1µg/ml and anti TGFβ3 1.4µg/ml). Following blocking, the cells were transfected by pGL3-p15-Lux wild type plasmid. After transfection the cell were incubated with exogenous recombinant TGF-β1/TGFβ3 (2 or 5ng/ml) for 24 hours in presence of the antibody. Background luciferase activity (empty vector) was subtracted from all data. Error bars indicate standard deviation of three independent preparations. After blocking endogenous TGFβ3, addition of TGFβ1 increases p15ink4b promoter activity in dose-dependent manner in MEE cells. After blocking endogenous TGFβ1, addition of TGFβ3 does not have significant effect on p15ink4b promoter activity.