SOPten flox/flox (KO) Pten flox/flox (WT) flox allele 6.0 kb. Pten. Actin. ! allele 2.3 kb. Supplementary Figure S1. Yanagi, et al.

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s1 A Pten flox/flox () SOPten flox/flox () flox allele 6. kb B Pten flox/flox () SOPten flox/flox () Pten Actin! allele 2.3 kb Supplementary Figure S1. Yanagi, et al.

A B BrdU BrdU positive cells ( ) 3 15 * Ks2 TUNEL DAPI TUNEL positive cells ( ) 8 4 Supplementary Figure S2. Yanagi, et al.

CD31 -SMA desmin GFAP Supplementary Figure S3. Yanagi, et al.

A B Cancer-free survival (%) 1 8 6 4 2 (n=48) (n=14) Adenocarcinoma n=13 Squamous cell carcinoma n=1 1 2 3 4 5 6 7 8 9 Time (weeks) p <.1 3 C Supplementary Figure S4. Yanagi, et al. D Number of tumors 2 15 1 5 * Mean tumor diameter (mm) 3 1.5 **

Supplementary Figure Legends Supplementary Figure S1. Generation of SP-C-rtTA/(tetO) 7 -Cre/Pten flox/flox (SOPten flox/flox ) mice. (A) Genomic Southern blot. DNA (2 µg) extracted from lungs of Pten flox/flox (E1-16) () and SOPten flox/flox (E1-16) () mice was digested with Hind III and hybridized to a previously described probe (24). Lungs from the majority of mice showed deletion of the Pten gene. The floxed (flox allele) and deleted (! allele) Pten alleles are shown. (B) Western blot analysis of Pten protein in lungs of 8- week-old (E1-16) and SOPten flox/flox (E1-16) () mice. Actin, loading control. Supplementary Figure S2. Increased cell proliferation in SOPten flox/flox (E1-16) lung (A) Enhanced proliferation. Left panel: BrdU-positive lung cells (arrows) in lungs of E19.5 (E1-16) and SOPten flox/flox (E1-16) () mice were counted 2 hr after BrdU administration to their dams. Bars, 5 µm. Right panel: data were expressed as the mean number of BrdU-positive lung cells ± SD/1!1 3 cells per mouse in 4 mice/group. *, p<.1, Student s t-test. (B) No effect on apoptosis. Upper left panels: TUNEL positive lung cells (arrows). Lower left panels: control DAPI staining. Bars, 5 µm. Right panel: data were expressed as the mean number of TUNEL-positive lung cells ± SD/1!1 3 cells/mouse in 4 mice/group. Supplementary Figure S3. Increase in number of myofibroblast precursors in the alveolar septa of SOPten flox/flox (E1-16) mice

Immunohistochemical analyses of "-SMA, a differentiation marker for smooth muscle cells and myofibroblasts, and desmin, a main component of the intermediate filaments of muscle cells, showed an increased number of "-SMA + desmin - cells in the septa of SOPten flox/flox (E1-16) () lungs. These cells are negative for GFAP, a marker protein for glial cells such as astrocytes. No obvious differences in the intensity or patterns of CD31 expression, a marker protein for vascular endothelial cells, were seen between (E1-16) and (E1-16) mice. Bars, 5 µm. Supplementary Figure S4. Spontaneous lung adenocarcinomas and urethaneinduced lung tumors in SOPten flox/flox (E1-16) mice (A) Gross and histological analyses of spontaneous tumors. Gross appearance of a lung in a 68-week-old (E1-16) mouse and representative lung carcinoma (arrow) in a lung of a 68-week-old SOPten flox/flox (E1-16) () mouse (upper panels). Lower left panel: spontaneous lung adenocarcinoma in a lung of a 72-week-old (E1-16) mouse, representative of tumors observed in 13/14 mice. Lower right panel: squamous cell carcinoma (1/14) in the lung of a 76-week-old (E1-16) mouse. Bars, 5 µm. (B) Kaplan-Meier curves showing cancer-free survival of untreated (E1-16) (n=48) and (E1-16) (n=14) mice. The statistical difference in survival was determined by the log-rank test. (C) Gross and histological analyses of urethaneinduced tumors. (E1-16) (n=26) and (E1-16) mice (n=18) were injected i.p. with urethane. After 2 weeks, tumors were counted and their diameters measured from

the lung surface. Upper panels: gross appearance of urethane-induced lung tumors (arrows) in (E1-16) and (E1-16) mice. Lower left panel: histology of a lung adenoma representative of those observed in both (E1-16) and (E1-16) mice. Lower right panel: histology of the one lung adenocarcinoma occurring in a (E1-16) mouse. Bars, 5 µm. (D) Numbers (left panel) and mean size (right panel) of urethane-induced tumors in (E1-16) and (E1-16) mice. The horizontal bars (left panel) represent the mean tumor numbers in the and groups. The right panel shows the mean diameter ± SD of tumors in the and groups. *, p<.5, **, p<.1, Welch s t-test.

Supplementary Table S1 Primers for PCR and RT-PCR experiments PCR SP-C-rtTA sense 5'-AAAATCTTGCCAGCTTTCCCC-3' antisense 5'-GACACATATAAGACCCTGGTCA-3' (teto) 7 -Cre sense 5'-TGCCACGACCAAGTGACAGCAATG-3' antisense 5'-AGAGACGGAAATCCATCGCTCG-3' Pten wt and flox allele sense 5'-CTCCCACCAATGAACAAACAGTC-3' antisense 5'-GTGAAAGTGCCCCAACATAAGG-3' K-ras, exon 1 sense 5'-ATGACTGAGTATAAACTTGT-3' antisense 5'-CTCTATCGTAGGGTCGTACT-3' K-ras, exon 2 sense 5'-ACTCCTACAGGAAACAAGT-3' antisense 5'-CTATAATGGTGAATATCTTC-3' K-ras, exon 1, sequence 5'-CTCTATCGTAGGGTCGTACT-3' K-ras, exon 2, sequence 5'-CTATAATGGTGAATATCTTC-3' RT-PCR Spry2 sense 5'-TGTGAGGACTGTGGCAAGTGC-3' antisense 5'-TTTAAGGCAACCCTTGCTGG-3' Shh sense 5'-GAAGATCACAAGAAACTCCGAACG-3' antisense 5'-TGGATTCATAGTAGACCCAGTCGAA-3' FGF7 sense 5'-CAAACGGCTACGAGTGTGAA-3' antisense 5'-TCCGCTGTGTGTCCATTTAG-3' FGF1 sense 5'-GAGAAGAACGGCAAGGTCAG-3' antisense 5'-GGAGGAAGTGAGCAGAGGTG-3' FGFR2 sense 5'-AAATACCAAATCTCCCAACC-3' antisense 5'-GCCGCTTCTCCATCTTCT-3' BMP4 sense 5'-AGGAGGAGGAGGAAGAGCAG-3' antisense 5'-TGTGATGAGGTGTCCAGGAA-3' Wnt7b sense 5'-CCCACATGACAGATGGACAG-3' antisense 5'-GGACCCTGAAGCAGGTATCA-3' Nkx2.1 sense 5'-AACCTGGGCAACATGAGCGAGCTG-3' antisense 5'-ATCTTGACCTGCGTGGGTGTCAGG-3' GATA6 sense 5'-CTTCTAATTCAGATGACTGC-3' antisense 5'-GCACGGAGGATGTGACTTC-3' Nfib sense 5'-CGAACATGGCACGAAAGAGA-3' antisense 5'-TGGACTGGATGGGTCATAAG-3' P3 sense 5'-GTGTCTCCACATGGTGCTTG-3' antisense 5'-GTGTCTCCACATGGTGCTTG-3' CBP sense 5'-CAGAGCGGATCATCCATGACTA-3' antisense 5'-GCTTCTCCATGGTGGCATAC-3' Tcf21 sense 5'-AGCTACATCGCCCACTTGAG-3' antisense 5'-CGGTCACCACTTCTTTCAGG-3' Cutl1 sense 5'-GGACGTAATGTCCGAAGATGC-3' antisense 5'-CTCAGAGGGCGAGGAAATTG-3' Hoxa5 sense 5'-GTGCCAATGTTGTGTGTT-3' antisense 5'-CATACATCACTGAACTGCGC-3'

Foxa1 sense 5'-GAAAGGCTAGCCAGCTAGAGG-3' antisense 5'-AGATGCAGCTGAGATTCGTG-3' Foxa2 sense 5'-CCCATTCCAGCGCTTCTC-3' antisense 5'-GTAATGGTGCTCGGGCTTC-3' GR sense 5'-CAAAGCCGTTTCACTGTCC-3' antisense 5'-ACAATTTCACACTGCCACC-3' Adam17 sense 5'-GCGGCGTCTCCTCATCCT-3' antisense 5'-TTATATTCTGCCCCATCTGTGTTG-3' HIF2" sense 5'-GGCGACAATGACAGCTGACAAGGAG-3' antisense 5'-GAGTGAAGTCAAAGATGCTGTGTC-3' enos sense 5'-AAGACAAGGCAGCGGTGGAA-3' antisense 5'-GCAGGGGACAGGAAATAGTT-3' GAPDH sense 5'-TGATGGGTGTGAACCACGAG-3' antisense 5'-GTCATTGAGAGCAATGCCAG-3'

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