Journal: Scientific reports

Article Title: A next-generation system for smoke inhalation integrated with a breathing lung-on-chip to model human lung responses to cigarette exposure.

doi: 10.1038/s41598-025-00438-z

Figure Lengend Snippet: Fig. 6. Differential effects of CS exposure on various co-culture dynamic models. (A) Overview of cells used in the dual culture “DC” (AXiAECs/hLMVECs), culture conditions (dynamic; ALI + Str) and treatment (CS exposure) performed. (B) Overview of cells used in the triple culture “TC pBDMs” (AXiAECs/hLMVECs/ pBDMs) and “TC THP1” (AXiAECs/hLMVECs/THP1), culture conditions (dynamic; ALI + Str) and treatment (CS exposure) performed. (C) TER (Ohm cm2) measured in “ALI + Str” conditions pre-exposure at 0 h and 4 h, 24 h and 48 h post-exposure to CS in DC (N = 3; n = 3/time-point), in TC pBDMs (N = 2; n = 3/time-point) and TC THP1 (N = 3; n = 3/time-point). Data are shown as violin plots, medians are indicated by black dotted lines. (D) The levels of IL-8 (pg/ml) in the supernatants collected from the cells were measured by ELISA in all the co-culture models. (E) Cytotoxicity was calculated from LDH release at 48 h time-point after CS exposure (N = 2; n = 3/time-point for all models). Data are shown as mean ± SEM.

Article Snippet: Human Lung Microvascular Endothelial Cells (hLMVECs) purchased from PromoCell were expanded in flasks using AX Endothelial Medium. hLMVECs were first seeded on the basolateral side of each membrane in the AX12 at a density of 106,000cells/cm2, followed by a 2-h incubation, and then AXiAECs were seeded apically at a density of 409,000 cells/cm2 on day 0 for all co-culture experiments.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: A next-generation system for smoke inhalation integrated with a breathing lung-on-chip to model human lung responses to cigarette exposure.

doi: 10.1038/s41598-025-00438-z

Figure Lengend Snippet: Fig. 7. Apical surfactant addition demonstrates potential protective effects against CS exposure. (A) Overview of cells used in the triple culture “TC THP1” (AXiAECs/hLMVECs/THP1), culture conditions (dynamic; ALI + Str) and treatment (Surfactant pre-treated followed by CS exposure) performed. (B) Representative immunofluorescence staining of CTRL ALI + Str (− Surfactant) cells (top row; scale bar 50 µm), CS exposed ALI + Str (-Surfactant) cells (second row; scale bar 50 µm), CTRL ALI + Str (+ Surfactant) cells (third row; scale bar 100 µm), CS exposed ALI + Str (+ Surfactant) cells (fourth row; scale bar 100 µm). Cells were stained for Zonula Occludens 1 (ZO-1, in green) and nuclei (Hoechst, in blue). (C) IL-8 concentration (pg/ml) measured from apical supernatants collected from the CTRL and CS exposed wells pre-treated with and without surfactant (+ Surf or -Surf) via ELISA assay (N = 2; n = 6/condition). (D) mRNA was harvested at 48 h time- point with and without surfactant (+ Surf or -Surf) post-exposure to CS. qRT-PCR study for inflammation (Interleukin 6, IL6; Interleukin 8, IL8; Tumor Necrosis Factor α, TNFα) and oxidative stress-related (Heme Oxygenase 1, HO-1; Human MutT Homolog 1, MTH-1) genes were conducted in all -Surf and + Surf CS exposed and CTRL samples (N = 2, n = 3). Data are shown as mean ± SEM.

Article Snippet: Human Lung Microvascular Endothelial Cells (hLMVECs) purchased from PromoCell were expanded in flasks using AX Endothelial Medium. hLMVECs were first seeded on the basolateral side of each membrane in the AX12 at a density of 106,000cells/cm2, followed by a 2-h incubation, and then AXiAECs were seeded apically at a density of 409,000 cells/cm2 on day 0 for all co-culture experiments.

Techniques: Immunofluorescence, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

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, Phospho-proteomics, 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