Nodal expression was directly inhibited using antisense Morpholino oligonucleotides

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1 Supplementary Note Rationale for Morpholino based inhibition of Nodal expression Nodal expression was directly inhibited using antisense Morpholino oligonucleotides (MO Nodal ) fluorescently labeled with FITC. The Morpholinos were designed to specifically bind the 5 mrna sequence of human Nodal, thereby blocking its translation. Given that the gastrulation phenotype in Nodal deficient mouse embryos can be suppressed by the inclusion of a small number of wild-type embryonic stem cells 1, we needed to ensure a complete knock out of Nodal synthesis. The FITC labeled Morpholinos served as a selection marker, thereby allowing us to efficiently purify the Nodal-deficient population of melanoma cells using fluorescence activated cell (FAC) sorting. Western blot analysis was used to determine the efficiency of this method. Reference List 1. Varlet,I., Collignon,J., & Robertson,E.J. Nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation. Development 124, (1997).

2 Supplementary Methods Cell Culture and Labeling The C81-61 cells were maintained in Ham s F10 (Invitrogen Life Technologies, Inc.) supplemented with 15% fetal bovine serum (FBS) (Gemini Bioproducts), 1X Mito+ (Collaborative Biomedical) and 0.1% gentamicin sulfate (Gemini Bioproducts). All of the other lines were maintained in RPMI 1640 (Invitrogen), supplemented with 10% FBS and 0.1% gentamicin sulfate (Gemini Bioproducts). The WM793 and 1205 Lu cell lines (a kind gift from Dr. Meenhard Herlyn s laboratory core, The Wistar Institute, Philadelphia, PA) were maintained in Melanoma Tu 2% medium (MCDB 153, Sigma Chemical Co.) supplemented with L-15 (Invitrogen), 5 g/ml bovine insulin (Sigma), 2% FBS, and 1.68 mm calcium chloride. The C8161 and C81-61 cells are respectively highly and poorly aggressive cutaneous melanoma cell lines derived from the same patient. The MUM-2B and MUM-2C cells are respectively highly and poorly metastatic uveal melanoma cells derived from the heterogeneous MUM-2 line, and 1205Lu was derived from an experimental lung metastasis of the WM793 cell line. Breast cancer cell lines were maintained in RPMI 1640 (Invitrogen), supplemented with 10% FBS and 0.1% gentamicin sulfate (Gemini Bioproducts) at 37ºC, 5% CO 2. Immunohistochemistry Formalin-fixed, paraffin-embedded archival tissue was obtained from patients with primary or metastatic cutaneous melanoma (Loyola University Chicago, IL). Immunohistochemical staining was performed on a HNS 710i Automated Immunostainer (Richard-Allan Scientific (RAS)) with the Multi-Species HRP/AEC Detection Systems. Following deparaffinization in xylene, ethanol degradation, and antigen retrieval with citrate buffer, four blocking steps were applied: 0.03% hydrogen peroxide, Avidin and Biotin blocks (Avidin/Biotin blocking kit, Vector Laboratories, Inc.), and a Serum-Free protein block. Anti-Nodal antibody (20 g/ml, R&D) was applied for 90

3 minutes. Slides were rinsed in TBS-T, incubated with biotinylated anti-goat IgG (2µg/ml, Vector Labs), washed with TBS-T and incubated with the streptavidin peroxidase reagent for 15 minutes. Color was produced with AEC (red) substrate (RAS) and counterstaining with Mayer s hematoxylin. Samples were dehydrated in reagent grade alcohol and cover slipped with permanent mounting medium. Negative control reactions were conducted with ChromPure Goat IgG (Jackson Labs), isotype matched and used at the same concentration as the Nodal antibody. RNA extraction and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated using TRIzol reagent (Invitrogen) in accordance to manufacturer instructions, and 1 µg was reverse transcribed using the Advantage PCR kit in accordance with the manufacturer s instructions (Clontech). Realtime PCR was performed on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) using TaqMan gene expression human primer/probe sets for Keratin 7 (Hs _m1) and VE-Cadherin (Hs _m1). cdna (5 µl), 1.25 µl 20X Gene Expression Assay Mix, and 12.5 µl 2X TaqMan Universal PCR Master Mix were mixed and 20 l was amplified with the following thermocycler protocol: 1 cycle at 50 o C for 2 min; 1 cycle at 95 o C for 10 min; and 40 cycles at 95 o C for 15 seconds/60 o C for 1 min. Target gene expression was normalized to the endogenous control gene GAPDH (GAPDH: F). Data was analyzed using Applied Biosystems Sequence Detection Software (Version 1.2.3). Statistical significance was determined using one way analysis of variance followed by the Holm-Sidak method for pairwise multiple comparisons. Differences were considered statistically significant at P < 0.05.

4 TUNEL assay Tunel assays to measure apoptosis were conducted as per instructions (Upstate).

5 Supplementary Table S1: Summary of Morphological Analyses of Zebrafish Injected with Human Melanoma Cells Ectopic outgrowth C8161 C81-61 C8161 MO Nodal (sorted) 90% n=240 0% n=170 0% n=20 C8161 MO Nodal (unsorted) 30% n=50 C8161 MO scramble 80% n=35 C8161 Lefty-1 16% n=43 Values represent the percentage of embryos displaying ectopic outgrowths and the n values are the number of embryos tested

6 Supplementary Table S2: Summary of Ectopic Gene Expression in Zebrafish Injected with Human Melanoma Cells ntl flh myod/krox20/pax2b ** gsc cyc sqt C % n=51 89% n=40 81% n=95 90% n=35 88% n=45 87% n=50 C % * ND ND ND ND ND n=165 C8161 Lefty-1 23% n=43 ND ND ND ND ND Values represent the percentage of embryos displaying ectopic gene expression and the n values are the number of embryos tested ND = not done * Only a few zebrafish cells expressed ntl ** These genes were tested simultaneously

7 Supplementary Table S3: Summary of Immunohistochemical Staining (IHC) and Western Blot Analyses of Nodal in Human Cutaneous Melanoma Specimens Nodal Staining (IHC) Case Type Nodal Signal (Western) Primary - Normal Skin - Primary - Normal Skin - Primary -* Normal Skin - Primary -* Normal Skin - Primary -* Normal Skin - Metastasis -* Normal Skin - Metastasis ++ Normal Skin - Metastasis + Normal Skin - Metastasis + Normal Skin - Metastasis - Melanocytes - Metastasis - Melanocytes - Metastasis - Melanocytes - Metastasis ++ Melanocytes - Metastasis ++ Melanocytes - Metastasis ++ Dendritic Cells - Dendritic Cells - ++ Represents strong positive staining for Nodal encompassing >75% of the tumor mass + Represents positive staining for Nodal encompassing >50% of the tumor mass or the detection of Nodal using Western Blot analyses -* Represents Nodal staining in a small subpopulation (<10%) of the tumor mass - Represents the absence of Nodal

8 Supplementary Table S4: Antibodies Utilized for Western Blot Analyses Antibody Concentration Company Polyclonal goat antimnodal Polyclonal rabbit anti- Nodal (H-110) Polyclonal rabbit antiphospho-smad-2 [psps 465/467 ] Polyclonal rabbit anti- Smad-2/3 Monoclonal mouse anti- Tyrosinase Polyclonal rabbit anti- Vascular Endothelial Cadherin Monoclonal mouse anti- Cytokeratin CK5 (anti- Keratin 18) Monoclonal mouse anti- Actin 150 ng / ml R&D Systems, Minneapolis, MN 1:500 Santa Cruz Biotechnology, Santa Cruz, CA 1:1000 Biosource, Camarillo, CA 1:1000 Upstate, Lake Placid, NY 1:500 Abcam Inc., Cambridge, MA 1:500 Cayman Chemical, Ann Arbor, MI 1:500 Sigma 1:5000 Chemicon International, Temecula, CA