ebm2 media (without growth factors)  (Lonza)

Journal: Scientific Reports

Article Title: Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

doi: 10.1038/s41598-017-14356-2

Figure Lengend Snippet: Ethanol conditioning increases endothelial cell-derived EV vascularization bioactivity. Gap closure by ( A ) HUVECs and ( B ) HDMECs was assessed following stimulation by 100 µg/ml EVs isolated from the same producer cell type in medium with the indicated ethanol (EtOH) concentrations (n = 3, *P < 0.05, **P < 0.01 vs. 0 mM EtOH condition); HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+). ( C ) Matrigel plugs injected into C57Bl/6 mice containing PBS as a negative control (−) or 100 µg HUVEC EVs from cells cultured with the indicated concentrations of EtOH in the media were removed 10 d after implantation and CD31+ cells were quantified using immunohistochemical staining (n = 6; **P < 0.01 compared to all other groups by one-way ANOVA). Data are presented as %CD31+ stained cells out of all cells counted for the gel sections from a given animal. ( D ) Representative gel images and immunohistochemistry sections from animals in the indicated groups are shown (n = 6).

Article Snippet: In gap closure experiments, EBM2 media (without growth factors) (Lonza) was used as the basal medium.

Techniques: Derivative Assay, Isolation, Incubation, Negative Control, Injection, Cell Culture, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

doi: 10.1038/s41598-017-14356-2

Figure Lengend Snippet: Ethanol (EtOH) regulates endothelial cell-derived EV microRNA content. ( A ) Luciferase expression in recipient HUVECs from a construct containing the 3′ untranslated region of CD34 was measured by bioluminescence imaging following stimulation by 100 µg/ml EVs from producer HUVECs cultured in the presence of the indicated concentrations of EtOH for 24 h (n = 4; **P < 0.01 vs. 0 mM EtOH condition, ## P < 0.01 vs. positive control (+) condition). Mock-transfected HUVEC not exposed to EVs were used as negative controls (−), while transfected HUVEC not exposed to EVs were used at the positive control (+). ( B ) Average whole miRNome array results comparing miR content of EVs derived from HUVEC cultured in the presence or absence of 100 mM EtOH for 24 h (n = 3). Red dots indicate upregulation of miRs and green dots indicate downregulation of miRs in HUVEC EVs based on producer cell EtOH exposure. Black dots indicate similar expression levels. ( C ) Expression levels of the indicated microRNAs (miRs) in endothelial cell EVs from producer cells cultured in the presence vs. absence of 100 mM EtOH for 24 h was determined by qPCR (n = 3). ( D ) Gap closure of HUVECs was assessed upon stimulated by EVs derived from HUVECs cultured in the absence (−EtOH) or presence (+EtOH) of 100 mM EtOH for 24 h following mock transfection or transfection by an antagomir to miR-106b (anti-miR-106b) or a scrambled oligo sequence (anti-miR-scr) (n = 3; ***P < 0.001, **P < 0.01 vs. EVs (−EtOH) group). HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+).

Article Snippet: In gap closure experiments, EBM2 media (without growth factors) (Lonza) was used as the basal medium.

Techniques: Derivative Assay, Luciferase, Expressing, Construct, Imaging, Cell Culture, Positive Control, Transfection, Sequencing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

doi: 10.1038/s41598-017-14356-2

Figure Lengend Snippet: Ethanol regulates endothelial cell-derived EV lncRNA content. ( A ) Expression levels of the indicated lncRNAs were assessed by qPCR in EVs from HUVECs cultured in the presence vs. absence of 100 mM EtOH for 24 h (n = 3; *P < 0.05). ( B – D ) HUVEC gap closure was assessed following 24 h stimulation by 100 µg/ml EVs from HUVECs cultured in the absence (−EtOH) or presence (+EtOH) of 100 mM EtOH for 24 h following transfection with a scrambled siRNA (scr) or siRNA specific to (B) HOTAIR, ( C ) MALAT1, or ( D ) both HOTAIR and MALAT1 (double transfection) (n = 4; ## P < 0.01 vs. – EtOH+ scr; **P < 0.01, ***P < 0.001 vs. +EtOH+ scr). HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+).

Article Snippet: In gap closure experiments, EBM2 media (without growth factors) (Lonza) was used as the basal medium.

Techniques: Derivative Assay, Expressing, Cell Culture, Transfection, Incubation, Negative Control