(A) Cells grown in monolayer were fixed and stained for surfactant protein-c (SPC,

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1 Supplemental Figure Legends Figure S1. Cell line characterization (A) Cells grown in monolayer were fixed and stained for surfactant protein-c (SPC, green) and co-stained with DAPI to visualize the nuclei. (B) Analysis of the p53 mutational status was carried out by PCR amplification, illustrating one band for the WT allele and a second for the mutant allele (MT). (C) To verify the presence of the Kras G12D mutation in each of the cell lines, purified DNA was used for Sanger sequencing, with the results displayed as chromatograms, illustrating that the cells possess one mutant allele and one wildtype. Figure S2. Karyotypic analysis of the cell lines. Metaphase spreads for each of the cell lines (393P, 393LN, 344P, and 344SQ) grown in culture were analyzed and display significant aneuploidy, with marker chromosomes displaying areas of translocation shown in red boxes for 393LN and 344P, and unidentified chromosomal fragments illustrated in 393LN and 344SQ. Figure S3. In vivo tumor growth and metastasis in syngeneic model. (A) Example of the gross pathology from an animal 5 weeks after subcutaneous injection of 1 million 344SQ cells, illustrating primary tumor formation (left, arrow), with significant neovascularization, and multiple metastases to the lung (right, arrows). (B) Hematoxylin and eosin stained tissue sections of a primary 344SQ syngeneic tumor (left) and a lung metastasis (right). Bar represents 100 µm. (C) Growth rates for each of the tumor types, including the parental 393P (non-metastatic) and 344SQ (metastatic) cells, and the 344SQ cells transfected with either the vector control or vector containing the mir-200b cluster. Dimensions (length and width) were measured at the time of necropsy and volume calculated as π(l)(w) 2 /6. The plot shows the volume standardized for the number of days between injection and necropsy. Figure S4. Q-PCR analysis of 344SQ cells grown in 2D versus 3D.

2 344SQ cells were grown as a monolayer on standard tissue culture dishes or in 3D on growth-factor-reduced Matrigel, followed by RNA purication. Syber green-based Q-PCR was used to measure the indicated markers from these samples and the values were normalized to the L32 gene levels measured on the same samples. Figure S5. Growth of 393P, 531P, and 1787A cells in 3-D Matrigel assay. (A) Morphology by contrast microscopy of 393P cells (non-metastatic) grown for 5 days on growth-factor-reduced Matrigel, or (B) after 12 days. (C) Morphology of 531P cells (metastatic) grown for 7 days on Matrigel, demonstrating sphere formation. (D-F) Confocal imaging of 393P cells grown on Matrigel for 10 days and stained for Topro3 to visualize the nuclei (blue) and the golgi marker GM130 (green, D), E-cadherin (green, E), or α6-integrin (red, F). Note that the round structures in D still have cells in the center and that the golgi marker (GM130) has random orientation in the various cells (arrows), similar to the α6-integrin staining in F, denoting a lack of epithelial polarity. Bar in confocal images represents 50 μm. (G) Morphology of 1787A cells (derived from an adrenal metastasis after 4 rounds of sequential passage of the 344P cells through syngeneic mice) grown on Matrigel for 7 days, demonstrating sphere formation. (H) Response of 1787A cells to TGFβ stimulation for 48 hours, demonstrating loss of organized morphology and invasion into the surrounding Matrigel. Figure S6. Differential TGFβ effects on metastasis-incompetent and metastasisprone tumor cells. (A) TGFβ1 levels from syngeneic tumor lysates, as measured by ELISA, and corrected for total protein concentration in each sample. Each bar represents a separate tumor, measured in triplicate at three different dilutions. (B) Contrast microscopy showing control 393P cells grown as a monolayer on standard tissue culture plastic, compared to the same cells treated for either 2 days (C) or 7 days (D) with 5 ng/ml TGFβ. (E) RNA was purified from the 393P cells grown for 7 days in TGFβ, as in panel D and then Syber

3 green-based Q-PCR was used to measure the indicated markers from these samples and the values were normalized to the L32 gene levels measured on the same samples. (F) Contrast microscopy showing control 344SQ cells grown as a monolayer on standard tissue culture plastic, compared to the same cells treated for either 2 days (G) or 7 days (H) with 5 ng/ml TGFβ. (I) RNA was purified from the 7 day treatment sample, as in panel H and Syber green-based Q-PCR was used to measure the indicated markers. The values were normalized to the L32 gene levels measured on the same samples. Figure S7. 3 UTR enrichment analysis of expression profiles from human NSCLC cell line panel. The correlation between mrna expression and motif content of mrna 3 UTRs in the panel of 40 NSCLC cell lines (as in Fig. 5A) was assessed using the program mireduce. We used log ratios of expression measurements for 15,962 genes and calculated correlations with the 7-mer motif content. Shown are the motifs meeting a nominal significance threshold of p < The 7-mer target site for mir-200b, mir-200c and mir-429 was the most highly enriched motif in genes with reduced expression in cell lines derived from primary tumors relative to those derived from metastases, observed in 2008 of the genes with a linear regression coefficient of and p < 10E -8. Table S1. Metastatic potential of cell lines in syngeneic mice. Table S2. mrna expression data for syngeneic tumors comparing 393P, 393LN and 344SQ. Table S3. GO Term analysis of mrna expression from syngeneic tumors. Table S4. microrna expression data for syngeneic tumors comparing 393P, 393LN and 344SQ. Table S5. Baseline mir expression levels for wildtype, vector and 200b stable transfectant cell lines.

4 Table S6. (A) Top genes upregulated in mrna expression data for mir-200b transfectant versus control vector transfectant. (B) Top genes downregulated in mrna expression data for mir-200b transfectant versus control vector transfectant. Table S7. Overlap in differential gene expression between 393P/344SQ pair and 344SQ_200b/344SQ_vector pair. (A) Genes downregulated with mir-200b expression. (B) Genes upregulated with mir0200b expression.

5 Figure S1. Characterization of murine cell lines. C 393P 393LN 344P 344SQ 344LN

6 Figure S2. Cytogenetic analysis of murine lung adenocarcinoma cell lines. 393P 393LN 344P 344SQ

7 Figure S3. In vivo growth & metastasis of murine lung adenocarcinoma cell lines. 45 A C mm 3 /day P 344SQ WT 344SQ.vector 344SQ.200b P 344SQ vector 200b B

8 Figure S4. Q-PCR analysis of markers in 344SQ cells from 2D vs 3D culture 7 6 2D 3D Normalized values 1 0 VIM CDH1 CDH2 DLG1 CRB3 CLDN3 ParD6B Scribble INADL ZEB1 SNAI2 SIP1

9 Figure S5. 3-D growth of 393P, 531P, and 1781A cell lines A D E B F C G H

10 Figure S6. Differential TGFβ effects on metastasis-incompetent and metastasis-prone tumor cells P 344SQ 0 TGFB levels (normalized to total protein) A B C D E Fold Change vs. Untreated F G H I 1 VIM ZEB1 CDH1 CDH2 CLDN3 INADL CRB CDH1 CLDN3 INADL CRB3 1 VIM ZEB1 CDH2 Fold Change vs. Untreated 0.5 0

11 Figure S7. 3 UTR enrichment analysis in human NSCLC cell lines mir-200bc, mir-429 (p<10-8)

12 Table S1. Metastatic Potential of Cell Lines in Syngeneic Mice a Route of Injection Local Metastasis Metastatic Sites 393 Primary SQ 7/10 b 0/10 c 393 LN Met SQ 6/6 0/6 344 Primary SQ 5/5 2/5 Lung 344 SQ Met SQ 13/13 13/13 lung, diaphragm, heart, pancreas, bone, kidney, muscle, subcutaneous 412 Primary SQ 2/3 2/3 Lung 531 Primary SQ 1/2 1/2 Lung a Tumor cells (10 6 per mouse) were injected subcutaneously, and mice were sacrificed upon demonstrating signs of morbidity or tumors reaching maximal allowable size. b Number of mice with tumors at injection site/total mice injected. c Number of mice with metastases at necropsy/total mice injected.

13 Table S5. Baseline mir expression levels for cell lines. mir c 200a 200b SQ WT SQ_Vector SQ_200b_ SQ_200b_