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: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Compared with CS, EGFL7 is highly expressed in OS. (A) Typical pathological features of OS and CS revealed by HE staining, tumor-like osteogenesis can be seen in OS and cartilage-like matrix in CS. (B,C) Immunohistochemical of EGFL7 protein expression in OS tissue ( n = 2) and CS controls ( n = 2), comparison of IHC results from different magnification (100 and 200 ×) and its significance. High expression of EGFL7 (++) was found in OS tissues but negative in CS, the arrow refers to the positive area. The semi-quantitative statistical analysis based on IHC results shows that there is a significant difference in the expression of EGFL7 protein between OS and CS. (D,E) Western blot results showed that EGFL7 protein was overexpressed in OS tissue compared with CS, Student's t -test shows that the difference between OS and CS has obvious statistical significance. The abundance of EGFL7 protein in OS tissue was similar to that of GAPDH protein. (F) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS tissues was significantly higher than that in CS tissues, and the difference was statistically significant. (G) Immunohistochemical results of chondrosarcoma and osteosarcoma showed that EGFL7 was highly expressed in osteosarcoma. (H) The immunohistochemical results of chondrosarcoma and osteosarcoma showed that CD34 was highly expressed in osteosarcoma, indicating that there were more abundant blood vessels in osteosarcoma. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Staining, Immunohistochemical staining, Expressing, Comparison, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: The primers used for Q-PCR

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques:

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Upregulation of EGFL7 expression in OS cell line compared with ECs and CS cell line. (A,B) Western blot results showed that EGFL7 protein was overexpressed in all OS cell lines and HUVECs compared to SW1353. Statistical analysis showed that the expression of EGFL7 in OS cell lines was significantly different from that in SW1353. Similarly, the expression of EGFL7 protein in HUVEC was significantly different from that in SW1353. (C) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS cell lines was significantly higher than HUVEC, there are also differences between HUVEC and SW1353, and the difference was all statistically significant. PCR and western blot experiments were all performed in triplicate. (D) The ELISA results of EGFL7 in cell culture medium also confirmed that there was indeed a high expression of EGFL7 protein in osteosarcoma cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Immunofluorescence technique was used to verify the expression of EGFL7 from histological and cytological aspects, respectively. (A) As seen in these representative images, the EGFL7 was labeled by special anti-EGFL7 antibody, and the cell nuclei were stained by DAPI. The merged image shows that green fluorescent signals for EGFL7 protein were expressed in both tumor tissues and vascular endothelial cells, and we have observed this expression from multiple samples and different optical multiples, respectively. White arrows indicate positive areas. (B) From the immunofluorescence results of OS cells, the EGFL7 protein was labeled by red fluorescent, the cell nuclei were stained by DAPI. The expression of EGFL7 protein in OS cells is located in the cytoplasm.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Immunofluorescence, Expressing, Labeling, Staining

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: The expression of EGFL7 deregulated in OS cells after cisplatin intervention. (A) Intervention of four osteosarcoma cell lines with different gradient concentration of cisplatin. Under light microscope, the number of cells changed significantly with different concentrations of cisplatin. The toxicity of cisplatin was detected by CCK-8 kit and the corresponding IC50 of various cells was calculated. (B) The results showed that cisplatin could significantly affect the proliferation rate of tumor cell lines. (C,D) Western blot was assessed to compare EGFL7 protein in OS cells before and after Cisplatin intervention, the results showed that the expression of EGFL7 protein deregulated after Cisplatin intervention. Paired T test showed that EGFL7 protein expression in OS cells was significantly deregulated after cisplatin intervention and the difference was statistically significant. (E) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS cell lines was significantly deregulated after cisplatin intervention and the difference was statistically significant. PCR and western blot experiments were all performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Concentration Assay, Light Microscopy, CCK-8 Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Chemotherapy deregulated expression of EGFL7 in osteosarcoma. (A) Samples obtained by biopsy before chemotherapy and by tumor resection after chemotherapy in the same patient. Pre-chemotherapy tissue specimens showed fish-like appearance, while post-chemotherapy tumor tissue showed obvious bone repair changes. (B,C) Immunohistochemical staining of the same patient's tumors before and after chemotherapy showed that there was almost no expression of EGFL7 in the tumors after chemotherapy (black arrows represent positive areas). The semi-quantitative statistical analysis based on IHC results shows that there is a significant difference in the expression of EGFL7 between pre-chemotherapy and post-chemotherapy. (D,E) Western blot results showed that EGFL7 protein was almost no expression in post-chemotherapy tissue compared with pre-chemotherapy tissue, Student's t -test shows that the difference has obvious statistical significance. (F) RT-PCR results showed that the transcription level of EGFL7 mRNA in post-chemotherapy tissue was significantly deregulated than that in pre-chemotherapy tissue, and the difference was statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction