Sox17 and Lung Progenitor Cell Figure 7. Sox17 interacts with Smad3 and inhibits TGF-b1/Smad3 transcriptional activity and Smad3 DNA binding. (A) MLE-15 cells were transfected with the TGF-b/Smad-responsive reporter 3TP-luciferase (3TP-Luc) and increasing amounts of vector, t-Sox17, or full length Sox17 in the presence or absence 2 ng/ml TGF-b1. Sox17 inhibited TGF-b1-mediated activation of 3TP-Luc. The graph represents average fold activity6standard deviation. Asterisks indicate statistical significance determined by Student’s t-test (p,0.05). (B–C) Lysates from MLE-15 cells expressing full length or mutant FLAG-Smad3 or Sox17-V5 (schematic representations) were incubated with GST only and GST-Sox17 (B) or GST- Smad3 (C), respectively. GST-pulldowns revealed an interaction between amino acids 129–359 of Sox17 and the linker region and MH2 domain of Smad3. MH1 and MH2, MAD homology domains; HMG, High mobility group. (D) MLE-15 cells were transfected with 3TP-Luc in the presence or absence Smad3 and wild type or mutant Sox17. Sox17 full length and C-terminal deletion inhibited Smad3-dependent transcriptional activity. Representative results are shown6standard deviation of the mean. Asterisks indicate statistical significance determined by Student’s t-test (p,0.05). (E) Sox17 antagonizes TGF-b1/Smad3-mediated repression of cyclin D1 promoter activity. MLE-15 cells were co-transfected with 2944/+187 cyclin D1- luciferase and Smad3 or Sox17 in the presence or absence of 5 ng/ml TGF-b1. The graph represents average fold activity6standard deviation. Pound signs and asterisk indicate statistical significance determined by Student’s t-test (p,0.05). (F) Sox17 blocks Smad3 DNA binding. MLE-15 cell were transfected with FLAG-Smad3 in the presence or absence of Sox17. After 24 h, cells were incubated with 5 ng/ml TGF-b1 for 8 h before harvesting. PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5711