Journal: Cancer Research

Article Title: Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

doi: 10.1158/0008-5472.can-10-0705

Figure Lengend Snippet: Figure 2. Survey of Notch receptors and Nodal expression in melanoma cell lines. A, RNA isolated from 2 poorly aggressive cell lines (UACC1273 and c81-61) and 4 aggressive cell lines (C8161, MV3, SK-MEL-28, and WM852) was assayed for gene expression of Notch1–4 and Nodal by semiquantitative PCR. GAPDH was a loading control. DNA contamination was excluded using no MMLV (not shown). B, protein lysates were analyzed for Notch receptor and Nodal proteins by Western blotting. Actin was a loading control. C, Nodal (green) and Notch4 (red) proteins were examined by confocal microscopy in C8161, MV3, and SK-MEL-28 cells (also Supplementary Fig. 1). Arrowheads in inset (*) denote regions of colocalization (yellow). DAPI (blue) marks cell nuclei. White bar represents 10 mm. D, cells positive for Nodal, Notch4, or both Nodal and Notch4 (Nodal þ Notch4) were independently counted using a 25 objective. For each category, mean þ SD was graphed as a percentage of total DAPI-positive nuclei (n ¼ 7). E, immunohistochemistry of Nodal and Notch4 on serial sections of a human melanoma tissue array. The number of tissue samples showing strong staining (>50%) was graphed as a percentage of total samples evaluated (for stage I–II, n ¼ 36; for stage III–IV, n ¼ 25). *, P < 0.05.

Article Snippet: C8161, MV3, and SK-MEL-28 cells seeded at confluence were antagonized with a goat anti-human Notch4 neutralizing antibody ((24); Supplementary Table 2) or goat whole molecule IgG (Jackson ImmunoResearch Laboratories) at 5 mg/mL.

Techniques: Expressing, Isolation, Gene Expression, Control, Western Blot, Confocal Microscopy, Immunohistochemistry, Staining

Journal: Cancer Research

Article Title: Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

doi: 10.1158/0008-5472.can-10-0705

Figure Lengend Snippet: Figure 3. Notch4 signaling regulates expression of Nodal. A, transfection of C8161 (left) and MV3 (right) cells with siRNA to Notch1 (siNotch1), Notch2 (siNotch2), Notch3 (siNotch3), Notch4 (siNotch4), or a negative control (siCON), followed by Western blotting for Nodal protein. Actin was a loading control. Relative Nodal expression was determined by densitometry (n ¼ 3). B and C, C8161, MV3, and SK-MEL-28 cells were treated with anti-human Notch4 neutralizing antibody or IgG. RNA was analyzed by real-time PCR for expression of Nodal mRNA (B). Protein lysates were analyzed for Nodal and actin (C), and relative Nodal expression evaluated by densitometry (n ¼ 3). *P < 0.05.

Article Snippet: C8161, MV3, and SK-MEL-28 cells seeded at confluence were antagonized with a goat anti-human Notch4 neutralizing antibody ((24); Supplementary Table 2) or goat whole molecule IgG (Jackson ImmunoResearch Laboratories) at 5 mg/mL.

Techniques: Expressing, Transfection, Negative Control, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Cancer Research

Article Title: Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

doi: 10.1158/0008-5472.can-10-0705

Figure Lengend Snippet: Figure 4. Inhibition of Notch4 activity limits cell proliferation and promotes apoptosis. C8161, MV3, and SK-MEL-28 cells were treated with anti-human Notch4 antibody or IgG. A–C, at 24-hour time points, cells were assayed for cell number (A), viability (B), and apoptosis (C) by flow cytometry. Cell number is shown as a percentage of the initial population, whereas viability and apoptosis are represented as the percentage of total cells. Plots represent the mean SD of 3 independent experiments done in triplicate; *, P < 0.05. D, Western blot analyses of HistoneH3 phosphorylation and PCNA expression (proliferation), and PARP cleavage (apoptosis) in C8161, MV3, and SK-MEL-28 cells. Actin was a loading control. Membranes were stripped between antibody detections. Western blots are representative of 3 experiments.

Article Snippet: C8161, MV3, and SK-MEL-28 cells seeded at confluence were antagonized with a goat anti-human Notch4 neutralizing antibody ((24); Supplementary Table 2) or goat whole molecule IgG (Jackson ImmunoResearch Laboratories) at 5 mg/mL.

Techniques: Inhibition, Activity Assay, Flow Cytometry, Western Blot, Phospho-proteomics, Expressing, Control

Journal: Cancer Research

Article Title: Regulation of the Embryonic Morphogen Nodal by Notch4 Facilitates Manifestation of the Aggressive Melanoma Phenotype

doi: 10.1158/0008-5472.can-10-0705

Figure Lengend Snippet: Figure 5. Inhibition of Notch4 signaling blocks vasculogenic mimicry and anchorage-independent growth in vitro in a Nodal-dependent manner. A, C8161 and SK-MEL-28 cells were seeded on 3D-collagen gel matrix and left untreated or treated with IgG, anti-Notch4 antibody, or anti-Notch4 antibody plus recombinant human Nodal (rNodal). White arrows indicate vascular-like network formation in untreated (far left), IgG-treated (center left), and anti-Notch4 þ rNodal-treated cultures (far right). Original magnification 100. B and C, relative colony formation in C8161 (B) and SK-MEL-28 (C) cells cultured on soft agar following pretreatment with IgG or anti-Notch4 antibody with or without rNodal. Graph indicates macroscopic colonies as a percentage (mean SD) of control.*, significant difference from untreated/IgG-treated cultures (P < 0.05); **, significant difference from anti-Notch4–treated cultures (P < 0.05). Graphs depict 1 of 3 representative experiments.

Article Snippet: C8161, MV3, and SK-MEL-28 cells seeded at confluence were antagonized with a goat anti-human Notch4 neutralizing antibody ((24); Supplementary Table 2) or goat whole molecule IgG (Jackson ImmunoResearch Laboratories) at 5 mg/mL.

Techniques: Inhibition, In Vitro, Recombinant, Cell Culture, Control