manufacturer’s protocol to examine expression of cell cycle-related genes in lungs from adult CCSPrtTA/tetO-Sox17 (n = 3) and CCSPrtTA (n = 3) mice maintained on Dox for 2 and 3 days. Reverse transcription reactions were performed with 2 mg of RNA and oligo(dT) primers using the SuperScript First-Strand Synthesis kit (Invitrogen). RT-PCR was performed using the following primers: p15 59-AGG CTT CCT GGA CAC GCT TG-39 and 59- AGA TGG GGC TGG GGA GAA AG-39; p21 59-CGA AAA CGG AGG CAG ACC AG-39 and 59-TCC TGA CCC ACA GCA GAA GAG G-39; p57 59-AAG CGA ACA GGC AGG CAA G-39 and 59-TAG AAG GCG GGC ACA GAC TC-39; Foxm1 59- CAC TTG GAT TGA GGA CCA CTT-39 and 59-GTC GTT TCT GCT GTG ATT CC-39; cyclin A2 59-ACC AAG AGA ATG TCA ACC CCG-39 and 59-GGT GAA GGC AGG CTG TTT ACT G-39; cyclin B1 59-AGG GTC GTG AAG TGA CTG GAA AC-39 and 59-TTG GGC ACA CAA CTG TTC TGC-39; cyclin D1 59-CAC AAC GCA CTT TCT TTC CA-39 and 59-GAC CAG CCT CTT CCT CCA C-39; cyclin E1 59-GCA GGC GAG GAT GAG AGC AG-39 and 59-ATA ACC ATG GCG AAC GGA ACC-39; L7 59-GAA GCT CAT CTA TGA GAA GGC-39 and 59-AAG ACG AAG GAG CTG CAG AAC-39. Plasmids The following expression constructs have been previously described: pCIG-Sox17 and pCIG-t-Sox17 (truncated Sox17) [13]; Sox17-V5 epitope-tagged constructs encoding for wild type Sox17 (1–419), a N-terminal deletion equivalent to the t-Sox17 isoform (129–419), C-terminal deletion prior to the transactivation domain (1–359), and a DNA binding domain point mutant (M76A); pGEX- mouse Sox17 encoding for glutathione S-transferase (GST)-Sox17 fusion protein [52]. The 3TP-luciferase TGF-b/Smad-responsive reporter plasmid [62] was obtained from Dr. Jeff Molkentin (Cincinnati Children’s Hospital Research Foundation), and human cyclin D1-luciferase promoter constructs, based on GenBank Accession number Z29078, have been previously described and were acquired from Dr. Karen Knudsen [63]. The pGEX-human Smad3 construct (Addgene plasmid 12630) encoding for GST-Smad3 was generated in the lab of Dr. Rik Derynck (UCSF) [64], and the FLAG-tagged Smad3 expression vectors were generated by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center) (FLAG-Smad3 (1–425), Addgene plasmid 14052; FLAG-Smad3 (1–145), Addgene plasmid 14965; FLAG-Smad3 (146–425), Addgene plasmid 14966; FLAG- Smad3 (220–425), Addgene plasmid 14967) [65,66]. Cell culture and Reporter Assays MLE-15 cells, an SV40 immortalized mouse lung epithelial cell line, were maintained in HITES medium [67]. Cells were seeded in 6-well culture plates at 16105 cells per well and transfected using FuGENE6 (Roche). Transfections included a pCMV-b- galactosidase expression vector as an internal control for transfection efficiency. Recombinant human TGF-b1 (R&D Systems) was added directly to the culture medium where indicated. Cells were harvested 24 h post-transfection and luciferase activity was measured using a Luciferase Assay System kit (Promega) and normalized to b-galactosidase activity. Exper- iments were performed three times in triplicate and statistical significance was determined by paired Student’s t-test. GST pulldowns The GST-Sox17 and GST-Smad3 fusion proteins (and GST only) were expressed in BL21 cells induced with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG). Cell pellets were resus- pended in lysis buffer (25 mM Tris, pH 8.0; 0.5 mM EDTA, 0.2 M NaCl; 1 mM DTT; 15 ml/ml protease inhibitor cocktail Sox17 and Lung Progenitor Cell (Sigma); 0.1 mM PMSF), sonicated, and GST and GST fusion proteins were purified using glutathione sepharose beads (Amer- sham). Sox17-V5 and FLAG-Smad3 constructs were expressed in MLE-15 cells. Whole cell protein lysates were harvested after 48 hours in cell lysis buffer (20 mM Tris, pH 8.0; 1 mM EDTA; 100 mM NaCl; 0.5% NP-40; 5 ml/ml protease inhibitor cocktail (Sigma); 0.1 mM PMSF), and precleared using glutathione sepharose beads. A 10% volume of the precleared lysates was retained for experimental inputs, and the remaining lysate was incubated with 4–8 mg of GST, GST-Sox17, or GST-Smad3 beads. Non-interacting proteins were removed by several washes (20 mM Tris, pH 8.0; 1 mM EDTA; 300–500 mM NaCl; 0.5% NP-40; 5 ml/ml protease inhibitor cocktail (Sigma); 0.1 mM PMSF) and samples were eluted by boiling in Laemmli sample buffer containing b-ME. Bound proteins were analyzed by SDS- PAGE and immunoblot using rabbit anti-FLAG (1:6000; Sigma) and mouse anti-V5-HRP (1:5000; Invitrogen) antibodies. Chromatin Immunoprecipitation MLE-15 cells were seeded at 16106 in 10 cm plates and co- transfected with FLAG-Smad3 and pCIG or pCIG-Sox17 using FuGENE6 (Roche). After 24 h, cells were switched to serum-free media and treated with 5 ng/ml rhTGF-b1 (R&D Systems) for 8 h. Crosslinking was performed by treating cells with 1% formaldehyde for 10 min at room temperature and was terminated by addition of 0.125 M glycine. After rinsing with cold 16PBS, cells from 3 plates per condition were pooled and incubated in hypotonic buffer (10 mM HEPES, pH 7.8; 10 mM KCl; 1.5 mM MgCl2; 0.5% NP- 40; 5 ml/ml protease inhibitor cocktail) for 30 min at 4uC. Pellets were collected by centrifugation and incubated in lysis buffer (50 mM Tris, pH8.0; 10 mM EDTA; 1% SDS; 5 ml/ml protease inhibitor cocktail) for 30 min at 4uC. Lysates were sonicated (Diagenode bioruptor) to shear DNA and cell debris was removed by centrifugation. Lysates were precleared with Protein A/G Plus beads (Santa Cruz), sonicated salmon sperm DNA, and BSA (1 mg/ml) for 2 h at 4uC. A 1% aliquot of precleared chromatin was removed for experimental inputs. Equal volumes of the remaining lysate were incubated with EZview Red anti-FLAG M2 beads (Sigma) or Protein A/G Plus beads and normal mouse IgG (1.5 mg) overnight at 4uC. Beads were washed once in dialysis buffer (50 mM Tris, pH8.0; 2 mM EDTA) and 4 times with wash buffer (100 mM Tris, pH8.0; 500 mM LiCl; 1% NP-40; 1% deoxycholic acid) followed by elution in 200 ml (50 mM NaHCO3; 1% SDS). DNA-protein complexes were reverse crosslinked by incubation in 0.3 M NaCl and RNase A at 65uC overnight. DNA was isolated by phenol/ chloroform extraction and ethanol precipitation and resuspended in 50 ml H20. Genomic DNA obtained from chromatin immunopre- cipitations was analyzed by real time PCR using Sybr Green (Roche) and an Opticon Monitor II system (MJ Research). Primers for the mouse p15 gene promoter were as follows: 59-CCA CCC CGC CTA TTT GTC-39 and 59-CCG TGA GAT TGC TAC AGC C- 39. Amount of DNA immunoprecipitated with FLAG and IgG beads was calculated based on threshold cycle [C(t)] using the DC(t) method and normalized to input samples. Results are expressed as fold enrichment of FLAG immunoprecipitated samples relative to IgG controls. Statistical significance was determined by paired Student’s t-test. Expression of Smad3 and Sox17 inputs was assessed by immunoblot using cell lysates from transfections done in parallel to chromatin immunoprecipitations. Supporting Information Figure S1 Sox17 increases proliferation of respiratory epithelial cells in the adult mouse lung. (A) Immunostaining for phospho- PLoS ONE | www.plosone.org 12 May 2009 | Volume 4 | Issue 5 | e5711