Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Isolation of extracellular vesicles from NTERA2 cells. ( a ) General outline of the differential centrifugation-based protocol used. ( b ) Protein immunoblotting with the antibody against the EV specific markers heat shock protein 70 (HSP70) and CD63, and the negative EV marker calnexin. Equal protein amounts (20 ug) of NTERA2 cell lysates (NT2), large extracellular vesicle (lEV), and small extracellular vesicle (sEV) preparations were analyzed. On the bottom: the filter after protein transfer and Red Ponceau protein revelation.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Isolation, Centrifugation, Western Blot, Marker

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Analysis of NTERA2 cell-derived extracellular vesicles by electron microscopy. Scanning electron microscopy ( a , b , d , e ) and cryogenic transmission electron microscopy ( c , f , g – l ) images of large and small extracellular vesicle (lEV and sEV) preparations. Magnification bars are reported in each image.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Derivative Assay, Electron Microscopy, Transmission Assay

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Extracellular vesicle quantification. ( a ) Nanoparticle tracking analysis (NTA) of NTERA2 extracellular vesicles. Representative image of size distribution of small extracellular vesicles or sEVs (red line) and large extracellular vesicles or lEVs (orange line). The inset histogram shows the mean ± standard mean error of sEV and lEV concentration. ( b , c ) Comparison of the concentration of particles measured by NTA ( b ) and the protein yield ( c ) in sEV and lEV samples isolated from NTERA2 and U87 cancer cells. The volume of resuspension is kept constant. To determine particle concentration, five and two different samples have been analyzed for NTERA2 and U87, respectively. For each sample, five experiment videos of 60 s duration were analyzed. To determine protein yield six and three different experiments have been analyzed for NTERA2 and U87, respectively. ns not significant, * p < 0.05; ** p < 0.01.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Concentration Assay, Comparison, Isolation

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Wound healing assay. ( a ) Images of U87 cells, both untreated and treated with NTERA2 large extracellular vesicles (lEVs) were taken at 0, 6, and 20 h after wound. The open area surrounded by a yellow line is the one calculated by the ImageJ software. ( b ) Relative wound healing area in the different conditions. Three independent experiments were performed, each one in duplicate. Data are shown as means ± standard error of mean. * p < 0.05; *** p < 0.001.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Wound Healing Assay, Software

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Cell proliferation and drug-sensitivity assay. ( a ) Cell proliferation assay. Absorbance of colorimetric assay of U87 cells after 48 and 72 h in absence (untreated) or presence of NTERA2 large extracellular vesicles (lEVs). No significant (ns) difference between the two samples was detected. ( b ) Temozolomide (TMZ) drug-sensitivity assay after 72 h of treatment, in combination or not with NTERA2 lEVs. Cell viability decreased with TMZ treatment, but no significant differences were detected between the effect caused by TMZ alone or in combination with NTERA2 lEVs. The results are shown as means ± standard error of mean from two independent experiments, each one in duplicate. ns not significant, ** p < 0.01.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Sensitive Assay, Proliferation Assay, Colorimetric Assay

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: CRIPTO protein distribution in NTERA2 cell line. ( a ) Representative image of NTERA2 cell culture. ( b ) Flow cytometry analysis of CRIPTO protein presence on the cell membrane of NTERA2 cells. Two populations of CRIPTO positive cells (high and low), exposing different levels of the protein on the membrane, were distinguished by the respective gates. ( c ) Distribution of the CRIPTO high and low populations. The results are a mean of three independent experiments. ( d ) Equal protein amounts (20 ug) of NTERA2 cell lysates (NT2), large extracellular vesicles (lEVs), small extracellular vesicles (sEVs), and supernatant at the end of the centrifugation procedure (Sup III) were immunoblotted with an antibody against CRIPTO or against the EV marker heat shock protein 70 (HSP70).

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Cell Culture, Flow Cytometry, Membrane, Centrifugation, Marker

Journal: Cancers

Article Title: A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

doi: 10.3390/cancers14153700

Figure Lengend Snippet: Rescue of U87 cell migration. ( a ) Cell migration at 0, 6, and 20 h of untreated U87 cells, U87 treated with NTERA2 large extracellular vesicles (lEVs) or NTERA2-lEVs previously incubated with anti-CRIPTO antibody (lEVs-α-Cripto) and a scramble antibody (lEVs-α-IgG), respectively. Open area is outlined by a yellow line and analyzed with ImageJ. ( b ) Relative wound healing area in the different conditions. Three independent experiments were performed, each in duplicate. Data are shown as means ± standard error of mean. Inhibitory effect of NTERA2-lEVs was partially rescued after 20 h when vesicles were pretreated with anti-CRIPTO antibody. Rescue was not observed when anti-IgG antibody was added. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The teratocarcinoma cell line NTERA2 was purchased from ATCC (ATCC-CRL-1973), whereas the glioblastoma cell line U87 was pursed from Merck (89081402, Kenilworth, NJ, USA).

Techniques: Migration, Incubation