primary human lung microvascular endothelial cells hlmvecs  (PromoCell)

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract alters PP2A activity and distribution within endothelial cells. (A) Human microvascular lung endothelial cells (HLMVEC) were grown to 80% confluence in 6-well plates and treated with 5% CSE for 10 min, 30 min, 1, 6, 12 and 24 h. PP2A activity assays were conducted on lysate protein isolated from the cells at baseline and after 5% CSE treatment. (B) PP2A activity assays were conducted on cytosolic, nuclear, cytoskeletal and membrane protein fractions of HLMVECs treated with and without 5% CSE for 24 h. (C) Immunoblots for the catalytic subunit of PP2A (PP2AC) were conducted on membrane fractions of HLMVECs treated with control media or 5% CSE for 24 h. Actin immunoblots were conducted on membrane fractions as a loading control.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Isolation, Membrane, Western Blot, Control

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: . PTP1B regulates PP2A activity in endothelial cells and cigarette smoke extract reduces PTP1B activity (A) PP2A activity assays were conducted on human lung microvascular endothelial cells grown to 70% confluence in 6-well plates and then treated with control, NSMase or PTP1B siRNA for 24 h. (B) PTP1B activity assays were conducted on human microvascular lung endothelial cells (HLMVEC) grown to 80% confluence in 6-well plates and treated with 5% CSE for 6–24 h.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Control

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract decreases calcium efflux and regulates PP2A activity in a calcium-dependent manner . (A) Human microvascular lung endothelial cells (HLMVEC) were grown to 80% confluence in 24-well plates and treated with control media or media with 1, 2, 5 or 10% CSE for 10 min. A calcium release assay was conducted on media collected from these cells. (B) PP2A activity assays were conducted on human microvascular lung endothelial cells (HLMVEC) grown to 80% confluence in 6-well plates and were treated with low calcium media that was switched to high calcium media or high calcium media that was switched to low calcium media after 5% CSE treatment.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Control, Release Assay

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract and PP2A inhibition alter endothelial cell permeability and occluding phosphorylation. (A) FITC permeability assays were conducted on human microvascular endothelial cells monolayers grown to 100% confluence on 3-µm pore collagen-coated PTFE membranes that were treated with 5% CSE for 2, 4, 6, 18 and 24 h. (B) FITC permeability assays were conducted on human microvascular endothelial cell monolayers grown to 100% confluence on 3-µm pore collagen-coated PTFE membranes that were treated with 1-µM Fostriecin for 24 h. (C) Occludin protein was immunoprecipitated from the endothelial cell lysates using a specific antibody. Immunoblots for p-threonine and total occludin were conducted on the immunoprecipitated protein.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Inhibition, Permeability, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: PP2A C silencing increases neutrophil adhesion in lung endothelial cells. Human lung microvascular endothelial cells were grown to 70% confluence in 6-well plates and then treated with control or PP2A C siRNA for 24 h. The wells were incubated with Calcein-labeled neutrophils and the fluorescence intensity of each well was then measured.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Control, Incubation, Labeling, Fluorescence