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

Journal: Journal of Cellular and Molecular Medicine

Article Title: Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

doi: 10.1111/jcmm.16022

Figure Lengend Snippet: AR increases the expression of miR‐7‐5p in SK cells. (A) Relative normalized expression of miRNAs by qRT‐PCR analysis in SK cells. oeAR significantly up‐regulates expression of miR‐7‐5p and shAR down‐regulates the expression of miR‐7‐5p in SK cells. (B) The effect of shmiR‐7‐5p on VM formation. The number of VM formations were counted and compared in SK and HA22T cells. (C) Quantification of VM formation in SK and HA22T cells. (D) The effect of shmiR‐7‐5p on expression of VE‐cadherin and Notch4 in SK and HA22T. Western blotting was performed as described in Materials and Methods. ## P < .01 compared with PWPI + PLV group. ** P < .01 compared with oeAR + PLV group

Article Snippet: Notch4 antibody for Western blotting analysis was purchased from Biorbyt.

Techniques: Expressing, Quantitative RT-PCR, Western Blot