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. Author manuscript; available in PMC: 2019 Jun 17.
Published in final edited form as: Cancer Res. 2007 Feb 15;67(4):1571–1579. doi: 10.1158/0008-5472.CAN-06-1680

Figure 5.

Figure 5.

TGF-β signaling was not restored and not required for Smad4 suppression of claudin-1 in SW480 cells. A, TGF-β signaling was not restored by Smad4 expression, but was restored by coexpression of Smad4 and wild-type TGF-β type II receptor (Rll) in Smad4-deficient SW480 cells. SW480, SW480control, and SW480Smad4 clones were transiently cotransfected with 0.05 μg p3TP-Lux, 0.04 phRL-TK, and 0.15 μg of a control or a type II receptor expression vector. Sixteen hours after transfection, cells were treated with 3 ng/mL TGF-β and allowed to grow for 48 h before luciferase reporter assay. Columns, luciferase activity mean of three independent experiments and represented in folds (TGF-β treatment versus vehicle treatment) in each individual line; bars, SD. *, P < 0.05. B, the effect of the inhibitor of the TGF-β receptor kinase, LY364947, on Smad4 inhibition of claudin-1 in SW480 cells. SW480, SW480control, and three SW480Smad4 clones were grown in the presence of vehicle or 5 Amol/L of LY364947 for 48 h and then lysed in a lysis buffer. Equal amounts of cell lysates were used for immunoblotting for Smad4, claudin-1, and β-actin. C, TGF-β signaling pathway was not required for Smad4 suppression of claudin-1 in HT29 cells. Smad4 expression restored the autocrine and paracrine TGF-β response, which could be blocked by the inhibitor of TGF-β receptor kinase LY364947. Cells were transiently cotransfected with 0.1 Ag p3TP-Lux, 0.01 Ag phRL-TK, and 0.4 Ag of a control vector or a Smad4 expression vector. Sixteen hours later, cells were treated, as indicated, with or without 5 Amol/L LY364947 and 3 ng/mL TGF-β and cultured for another 48 h before reporter assays. Bars, SD. *, P < 0.05. D, the inhibitor of TGF-β receptor kinase, LY364947, did not abrogate Smad4 suppression of claudin-1 in HT29 cells. Cells were infected with β-galactosidase or Smad4 adenoviruses at the indicated multiplicity of infection for 4 h and then cultured for 72 h in the presence or absence of 5 Amol/L LY364947 before being lysed for immunoblotting for Smad4, claudin-1, and β-actin.