complete medium egm-2 bulletkit  (Lonza)

Journal: International Journal of Molecular Sciences

Article Title: The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo

doi: 10.3390/ijms22073672

Figure Lengend Snippet: ( A–C ) . Western Blotting analysis of cytoplasm and nuclear fraction of nucleophosmin (NPM) in human vein endothelial cells (HUVEC) after serum starvation. ( A ) Representative blot displaying the shuttle of NPM. Lamin B and α-tubulin have been employed as nuclear and cytoplasmic protein, respectively. ( B ) The densitometry analysis shows that NPM translocates from nucleus to the cytoplasm after serum deprivation (w/o Serum) but not in the presence of serum (w/Serum). Data are the results of 3 independent experiments. * p < 0.05, *** p < 0.001. ( C ) Immunofluorescent staining of HUVEC confirming a delocalized NPM in the cytoplasm in the absence of serum (w/o Serum) but not in presence of serum (w/Serum). NPM and DAPI stain green and blue, respectively. Scale bar is displayed.

Article Snippet: Cells were cultured in HUVEC complete growth medium (EGM TM -2 Endothelial Cell Growth Medium-2 BulletKit TM Lonza, Cat. N. CC-3162.

Techniques: Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo

doi: 10.3390/ijms22073672

Figure Lengend Snippet: ( A , B ). Secretion of NPM by HUVEC in serum starvation conditions and in absence of cell necrosis. ( A ) Active release of NPM into the microenvironment of HUVEC according to a time course (6, 18, 24 h) of serum deprivation (w/Serum) and measured by ELISA, showing a gradual increase of the protein in the culture media of endothelial cells at 18 and 24 h compared to 6 h. Data are the results of 4 independent experiments (technical duplicates). * p < 0.05, ** p < 0.01. ( B ) Concentration of the enzyme lactate dehydrogenase (LDH) displaying the absence of cell necrosis in both conditions (with Serum, w/Serum and without Serum, w/o Serum). Data are the results of 3 independent experiments (technical duplicates). O.D. optical density.

Article Snippet: Cells were cultured in HUVEC complete growth medium (EGM TM -2 Endothelial Cell Growth Medium-2 BulletKit TM Lonza, Cat. N. CC-3162.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo

doi: 10.3390/ijms22073672

Figure Lengend Snippet: ( A – C ). Biological effects of exogenous NPM on endothelial cells. ( A ) The graph shows that the exogenous recombinant NPM (rNPM 200 ng/mL, w/rNPM) is able to foster migration of HUVEC. The 10% Fetal Bovine Serum (FBS) and the EBM TM -2 have been used as positive and negative stimulus, respectively (Pos Ctl, positive control; Neg Ctl, negative control). Below the graph are representative images of migrated cells. Magnification 4X. Data are the results of 5 independent experiments (technical duplicates). * p < 0.05, ** p < 0.01. ( B ) Proliferation assay (MTS assay) showing that the increased migration of HUVEC upon rNPM treatment is not associated to an enhancement of the proliferation up to 96 h. Data are the results of 4 independent experiments (technical duplicates). * p < 0.05. Complete medium has been used as the positive control (Pos Ctl). O.D., optical density, normalized vs. time 0. ( C ) FACS Analysis displaying scatter plots (top panel) with gating strategy to analyze only viable cells for ICAM-1 and VCAM-1 expression in the presence (w/rNPM) or in the absence of rNPM (w/o rNPM) treatment. Quantification plot (Bottom panel) showing that rNPM can significantly increase the percentage of positive HUVEC for ICAM-1 but not for VCAM-1. Data are the results of 4 independent experiments. ** p < 0.01.

Article Snippet: Cells were cultured in HUVEC complete growth medium (EGM TM -2 Endothelial Cell Growth Medium-2 BulletKit TM Lonza, Cat. N. CC-3162.

Techniques: Recombinant, Migration, Positive Control, Negative Control, Proliferation Assay, MTS Assay, Expressing