Supplemental Table S1

Transcription

1 Supplemental Table S. Tumorigenicity and metastatic potential of 44SQ cell subpopulations a Tumorigenicity b Average tumor volume (mm ) c Lung metastasis d CD high /4 8. 8/ CD low /4 6./ a Mice were injected subcutaneously with, tumor cells and sacrificed after 6 weeks b number of mice with palpable tumors at injection site relative to total number of mice injected c bi-dimensional measurements d number of mice with visible lung metastases at time of necropsy relative to the total number of mice bearing visible subcutaneous tumors Supplemental Table S

2 Supplemental Table S. Effect of Jagged and Jagged knockdown on 44SQ and 44P tumor growth and metastasis a Cell Lines Tumorigenicity b Lung Metastasis c 44SQ-scr for Jag 8/8 8/8 44SQ-Jag shrna 6/8 p =. d /6 p =.55 44SQ-scr Jag 5/5 4/5 44SQ-Jag shrna /5 p =. / p <. 44P-scr 8/ 5/8 44P-Jag shrna 9/ p = /9 p =.4 a Mice were injected subcutaneously with the indicated tumor cells and sacrificed after 6 weeks. b number of mice with palpable tumors at injection site relative to total number of mice injected. c number of mice with visible lung metastases at time of necropsy relative to the total number of mice bearing visible subcutaneous tumors d incidence in controls versus Jagged shrna transfectants (One-sided Fisher exact test) Supplemental Table S

3 Supplemental Table S. Effect of Gata knockdown on 44SQ and 44P tumor growth and metastasis a Cell Lines Tumorigenicity b Lung Metastasis c 44SQ-scr 5/5 5/5 44SQ-Gata shrna 5/5 p =. d /5 p =.4 44P-scr 8/ 5/8 44P-Gata shrna 9/ p = /9 p =.4 a Mice were injected subcutaneously with the indicated tumor cells and sacrificed after 6 weeks b number of mice with palpable tumors at injection site relative to total number of mice injected c number of mice with visible lung metastases at time of necropsy relative to the total number of mice bearing visible subcutaneous tumors d incidence in controls versus Gata shrna transfectants (One sided Fisher exact test) Supplemental Table S

4 Supplemental Table S4. Correlation of GATA with EMT in CD high and CD low human lung adenocarcinomas a CDH c VIM c ZEB c N d R e P e R P R P b CD high <..46 <. Chitale dataset b CD low Tomida dataset CD high CD low a Human lung adenocarcinomas from two independent patient cohorts (Chitale et al., 9, and Tomida et al., 9) b CD high or CD low defined on the basis of PROM expression above or below the median value, respectively c GATA was correlated with CDH, VIM, or ZEB by performing two sided Spearman's rank correlation coefficient analysis d Cohort sample size e Spearman s R and P values Supplemental Table S4

5 A B % CD staining Cells p=.9 9P 44SQ CD.7% Supplemental Figure S. CD expression in murine lung adenocarcinoma cells. (A) CD is expressed more frequently in metastasis-prone 44SQ than it is in non-metastatic 9P lung adenocarcinoma cells. CD expression was detected in monolayer cultures by anti-cd immunofluorescent staining. Positive cells were counted and expressed as the number of positive cells per high powered field. (B) Flow cytometry to isolate CD high and CD low cell fractions. CD high fraction (P8) isolated from 44SQ subcutaneous tumors. Percentages of total cells subjected to flow sorting that were CD high are indicated (upper left corner of box).

6 CD Jag CD / Jag CD / Jag / DAPI Supplemental Figure S. CD high 44SQ cells are heterogeneous in Notch ligand expression. Illustrated are CD-expressing tumor cells that either do (middle row) or do not (bottom row) co-express Jagged. Immunofluorescent staining of a subcutaneous 44SQ tumor to detect Jagged (red) and CD (green), which were merged. Sections were co-stained with DAPI (blue). Scale bar in lower left corner of each panel indicates μm. Top row (4x), middle and bottom rows (x).

7 A Scr Gata KD p=.6 p<. p=.4 p<. p= Gata Gata Gata4 Gata5 Gata6 B Vec Gata cdna p<. p<. p<. p=.44 p< Gata Gata Gata Gata4 Gata5 Supplemental Figure S. Gata regulates the expression of Gata, Gata, and Gata5. Quantitative RT-PCR was performed to determine the relative mrna levels of other Gata family members in (A) 44SQ cells stably transfected with control shrna (Scr) or Gata shrna (Gata KD) and (B) Gata KD cells stably transfected with vectors that are empty (vec) or express Gata cdna. Note that the expression of Gata, Gata, and Gata5 is suppressed by Gata knockdown and restored in Gata KD cells by forced Gata expression. Values represent means (±S.D.) of replicate (triplicate) samples.

8 A Vector Jag KD p<. p<. p<. p<. p<. p< Jag Gata mir-a mir-b mir-c mir-49 B Vector Gata KD p<. p<. p<. p<. p< Gata mir-a mir-b mir-c mir-49 Supplemental Figure S4. Jag and Gata suppress expression of mir- family in 44P lung adenocarcinoma cells. Gene expression was analyzed in 44P cells stably transfected with shrnas against Jagged (A) or Gata (B). Values represent means (±S.D.) of replicate (triplicate) samples.

9 Scr JAG KD p <. p =. p =. p =. p =.7 p < JAG GATA mir-4 mir-a mir-b mir-c Supplemental Figure S5. Jagged increases GATA and suppresses mir- in a human lung cancer cell line. HCC5 cells were transiently transfected with control (scr) or Jagged (JAG KD) shrnas. Quantitative RT-PCR was performed to determine the relative expression of Jagged, GATA, and mir- family members. Values represent means (±S.D.) of replicate (triplicate) samples.

10 Supplemental Figure S6. mir- suppression by Gata requires the putative Gata binding site. Reporter assays performed on MCF-7 cells co-transfected with Gata (black bars) or control (white bars) expression vectors and pgl-based reporters containing nothing (pgl), wild-type mir-b promoter sequences from - to +9 (), the promoter fragment containing mutations in the E boxes (E-box mut) from - to -5 (CACCTG to CACCGG for each E-box), or the promoter fragment containing mutations in the putative Gata binding site (Gata mut) from -79 to -7 (GAGATTGGC to GACCTTGGC). Values were normalized based on renilla luciferase and expressed as the means (±S.D.) of replicate (triplicate) wells relative to cells co-transfected with empty vector, which were set at..

11 A B Wild type GFP + 44SQ-GFP % GFP +.% C CD low CD high GFP + Jag Gata Supplemental Figure S7. Jagged and Gata are co-expressed in early metastases. (A) Gata detected in metastases but not primary lung tumors in Kras LA/+ ; p5 R7HΔG/+ mice. Gata immunofluorescent staining (top panels) of a primary lung tumor (left), cardiac metastasis (middle), and hepatic metastasis (right). Gata-positive cells are green. Adjacent tissues sections stained with hematoxylin and eosin (bottom panels). Bars: μm. (B) Syngeneic mice were subcutaneously injected with or without (wild type) GFP-expressing 44SQ cells (44SQ-GFP) for weeks, and tumor cells of early lung metastases were analyzed and collected by flow cytometric sorting for GFP + cells from mouse lungs, which represents.% of total lung cells. (C) Quantitative PCR were performed to determine the relative expression of Jagged and Gata in CD low, CD high, and GFP + early metastatic 44SQ cells from (B).