Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: COPD epithelia demonstrate cellular plasticity with worsened monolayer barrier integrity, lower monolayer height and increased expression of mesenchymal markers. (A) FITC–dextran permeability is greater in gender- and age-matched COPD epithelial samples (CHBE) than NHBE. (B) Transepithelial resistance (TEER) is lower in gender and age-matched CHBE than NHBE. (C) Immunofluorescence staining of apical polarity (PKC-ζ, green) and basal polarity (Na + /K + -ATPase, red) shows preservation of polarity in both non-diseased epithelia (NHBE) and CHBE, although the CHBE has a much shorter monolayer height. Images representative of three donors per group (two inserts per donor). (D) The height of pseudostratified epithelium of the NHBE is greater than that of CHBE. (E­–L) There is decreased mRNA expression of epithelial (CDH1) in CHBE compared to age- and gender-matched NHBE (E), and an increase in mRNA expression of mesenchymal markers (CDH2, VIM, SNAl1, SNAl2, TWIST2, ZEB1 and ZEB2) relative to GAPDH in CHBE as analyzed by qPCR (F–L). Data is generated from three donors per group (age- and gender-matched and one to three inserts per donor). Data is expressed with median bars. Statistics determined by Mann–Whitney test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Permeability, Immunofluorescence, Staining, Preserving, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Repetitive exposure to cigarette smoke drives epithelial plasticity with worsened monolayer barrier integrity, lower monolayer height and increased expression of mesenchymal markers. (A,B) In CS-exposed NHBE cells TEER is lower (A), and FITC–dextran permeability is greater (B) than in NHBE cells exposed to air. (C) CS exposure in NHBE cells results in a quantitative decrease in the height of pseudostratified epithelium as compared to air-exposed NHBE. (D) Immunofluorescence staining shows preservation of apical-basal polarity (apical, PKC-ζ, green; basolateral, Na + /K + -ATPase, magenta) taken with a 40× oil objective. Scale bar: 25 µm. (E) Scanning electron microscopy shows preservation of specialized apical structures such as cilia (images taken at 2.5× and 800×). Scale bars: 2 µm, 10 µm. (F) Transmission electron microscopy images showing preservation of the 9+2 ciliary structure in despite CS exposure. Images in D–F representative of three donors per group (two inserts per donor). (G–M) Basal mRNA expression of the epithelial marker CDH1 is decreased with CS exposure (G), and mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) relative to GAPDH is increased (H–M) with CS exposure than in NHBE cells exposed to air as analyzed by qPCR. Data is generated from three donors (two to three inserts per donor) with graphs showing median bars. Statistics determined by Mann–Whitney test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Permeability, Immunofluorescence, Staining, Preserving, Electron Microscopy, Transmission Assay, Marker, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Comparative analysis of epithleial plasticity of pEMT in primary non-diseased human bronchial epithelia. (A) NHBE cells undergoing pEMT induced by treatment with 2× EMT inducer supplement (EMT supp) display increased FITC–dextran permeability compared to control PBS-treated (PBS Ctrl) cells. (B) NHBE cells undergoing pEMT display decreased TEER. (C) NHBE cells undergoing pEMT have a lower height of pseudostratified epithelium. (D) There is an expected loss of apical-basal polarity in the cells undergoing EMT as determined by immunofluorescence (basolateral, Na + /K + -ATPase, magenta). Taken with a 40× oil objective. Scale bars: 25 µm. (E) There is a significant loss of specialized apical structures in cells undergoing pEMT such as cilia, as assessed using scanning electron microscopy. Images taken at 2.5× and 800×. Scale bars: 2 µm, 10 µm. (F) Using transmission electron microscopy, cilia were identified in NHBE treated with PBS Ctrl (top panel); however, there were no cilia evident to capture in the EMT sample, highlighting the loss of specialized apical structures. Images in D–F representative of three donors per group (two inserts per donor). (G) Representative blots (top panel) and quantification (bottom panel) showing decreased E-cadherin expression in cells treated with EMT Supp compared to those treated with PBS Ctrl as detected by western blotting. β-actin levels are shown as a loading control. Blot representative of four inserts from one donor. (H–N) Basal mRNA expression of the epithelial marker CDH1 is lower in pEMT-induced NHBE cells (H), and levels of mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) relative to GAPDH are increased (I–N) with treatment with EMT Supp were compared to PBS Ctrl, as analyzed by qPCR. Data is generated from three donors (two to four inserts per donor). Data is expressed with median bars. Statistics determined by Mann–Whitney test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Permeability, Control, Immunofluorescence, Electron Microscopy, Transmission Assay, Expressing, Western Blot, Marker, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Epithelial plasticity is associated with increased cell velocity. (A) Representative heat maps of cell velocity of NHBE exposed to air or cigarette smoke (CS) and CHBE exposed to clean air for 10 days, showing increase in velocity in injured and diseased cells. (B–D) CHBE typically have increased mean cell velocity (B) with CHBE having an altered distribution of spatial autocorrelation function (C) and have a higher correlation length (D) as compared to age- and gender-matched NHBE epithelia. (E–G) CS-exposed NHBE have a higher mean cell velocity (E) with an altered distribution of spatial autocorrelation function (F) and increases in the correlation length (G) in air-exposed NHBE. (H–J) Cells undergoing pEMT induced by treatment with EMT inducer supplement (EMT supp) show increased mean cell velocity (H) with an altered distribution of the spatial autocorrelation function (I) and increased correlation length (J) compared to age- and gender-matched NHBE treated with PBS Ctrl. (K) Representative images of three donors (three inserts per donor) used for cell shape factor quantification in NHBE exposed to air or CS and CHBE. (L) Comparison of quantified cell shape among NHBE exposed to air or CS and CHBE demonstrated an increased cell shape factor in CS-exposed and CHBE cells. Data is generated from three donors (three inserts per donor) and the mean±s.d. is given underneath in L. Graphs are generated with median bars for cell velocity and correlation length. Statistics determined by Mann–Whitney test (B,D,E,G,H,J) or Kruskal–Wallis test followed by Dunn's multiple comparison test (L), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Comparison, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Both cigarette smoke exposed non-diseased and COPD-derived epithelia have an increased fraction of polymerized actin. (A) Representative western blot of globular (G) actin and filamentous (F) actin (top panel) and the quantification of polymerized actin (bottom panel) demonstrates that there is a higher polymerized fraction of actin in CS-exposed cells and in cells treated with the polymerizing agent (JaspA), while treatment with actin depolymerizing agent (LatA) reduces the fraction of polymerized actin. Data is representative of three inserts from one donor. (B) Representative western blot of G and F actin (top panel) and quantification of polymerized actin (bottom panel) in age and gender matched cells demonstrate an increase in polymerized fraction of actin in CHBE. Data is representative of three donors (one to two inserts per donor). (C,D) Representative image of immunofluorescence demonstrates increased fluorescence of phalloidin in (C) NHBE exposed to CS and (D) CHBE compared to their respective controls. Data is representative of two donors (two inserts per donor) with images taken under identical exposures. Scale bars: 25 µm. (E) Cells undergoing pEMT do not have an increased fraction of polymerized actin, as shown by the representative western blot of G-actin and F-actin (top panel) and quantification of polymerized actin (bottom panel) of in NHBE treated with PBS Ctrl and EMT Supp (2× EMT inducer supplement). Data is representative of four inserts from one donor. (F) A decrease in phalloidin is seen in sections of cells undergoing pEMT (representative image of immunofluorescence staining of phalloidin in NHBE treated with PBS Ctrl and EMT Supp; images taken under identical exposures). Data is representative of two donors (two inserts per donor). Scale bars: 25 µm. Polymerized actin is calculated as the summation of F-actin to total actin (G-actin+F-actin). Statistics determined by Mann–Whitney test (B,E) or Kruskal–Wallis test followed by Dunn's multiple comparison test (A), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Fluorescence, Staining, MANN-WHITNEY, Comparison

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Inhibitors of actin polymerization improve monolayer integrity and reverse plasticity. (A–C) LatA (an actin polymerization inhibitor) and U0126 (a MAPK inhibitor) are the only drugs which reduced cellular velocity (A), TEER (B) and correlation length (C) on the CS-exposed epithelia [cells treated with pathway modulators such as LatA, the actin-polymerizing agent JaspA, U0126, the TGFβ1 neutralizer (1D11), or an antagonist of Wnt signaling pathway (DKK1) and compared to vehicle control]. Data is representative of two donors (three to six inserts per donor). (D) None of the inhibitors restored the CS-induced decrease in basal mRNA expression of the epithelial marker CDH1 . (E–J) The levels of the mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) were significantly reduced relative to GAPDH in CS-exposed epithelia treated with several pathway modulators (LatA, U0126, 1D11 and DKK1) as analyzed by qPCR. (K) LatA and U0126 treatment reduced mean square displacement (MSD) in CS exposed epithelia. Data is representative of three to six inserts from two donors. (L–O) LatA and U0126 treatment improved TEER in CHBE cells (L), LatA and U0126 treatment decreased cellular velocity in COPD epithelia (CHBE) (M), LatA and U0126 treatment reduced correlation length in CHBE cells (N), and LatA and U0126 treatment improved the height of pseudostratified epithelia on COPD epithelia (CHBE) (O). (P) Representative DIC image from three inserts from one donor taken with a 40× oil objective of CHBE treated with or without LatA or U0126 showing improved monolayer height. Scale bars: 25 µm. (Q) LatA and U0126 treatment reduced the MSD with time in CHBE cells approaching levels similar to that of NHBE. Of note, there is not a significant increase in MSD with time in pEMT-induced NHBE. Data is representative of nine inserts, except for cellular velocity and correlation length (six inserts) and height of pseudostratified epithelia (three inserts) from one donor of CHBE. Graphs are shown with median bars. Statistics determined by Kruskal–Wallis test followed by Dunn's multiple comparison test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Control, Expressing, Marker, Comparison

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Restoring cofilin-1 improves airway epithelial integrity. (A) Age- and gender-matched CHBE cells have lower cofilin-1 expression (representative blot, left panel and quantification, right panel) as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of four donors, one to two inserts per donor). (B) There is reduced cofilin-1 expression as seen by immunofluorescence. Representative image from four donors (two inserts per donor) of cofilin-1 (red) and DAPI (blue) in NHBE, CS-exposed NHBE and CHBE epithelia. Taken with a 40× oil objective under identical exposures. Scale bars: 25 µm. (C) Despite having lower protein expression, CHBE express higher mRNA levels of CFL1 (encodes for cofilin-1) as analyzed by qPCR. Data is representative of three to four donors (three inserts per donor). (D) CS-exposure reduces cofilin-1 expression (representative blot, left panel and quantification, right panel) as detected by western blotting and GAPDH levels are shown as a loading control. Data is representative of three inserts from one donor. (E–G) mRNA expression of several actin-binding proteins were altered, with increases in ARPC2 , ARPC3 and PFN1 as analyzed by qPCR. Data is representative of three to four donors (three inserts per donor). (H) Cells treated with EMT Supp to induce pEMT do not have decreased cofilin-1 expression (representative blots, left panel and quantification, right panel) as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of four inserts from one donor). (I–U) NHBEs were transduced with adenovirus to knockdown or overexpress cofilin-1 [Ad-GFP (GFP), Ad-GFP-U6-h-CFL1-shRNA (KD) and Ad-GFP-h-CFL1 (OE) at 1×10 10 PFU/ml]. (I) Transfection of GFP-tagged control virus, CFL1 shRNA virus, and CFL1 overexpression virus had an efficiency of ∼40–60%, as visualized by GFP fluorescence. Taken with a 20× objective. Scale bars: 100 µm. (J,K) Representative blot (left panel) and quantification (right panel) of cofilin-1 normalized to GAPDH as loading control as detected by western blotting (J), and mRNA expression of CFL1 (encodes for cofilin-1) indicating ∼50% knockdown and ∼2-fold overexpression (K). Data is representative of two to three inserts from two donors for western blotting and three inserts from one donor for qPCR. (L) Representative blot of NHBE transduced with GFP-tagged control virus (GFP), and CFL-1 shRNA virus GFP (KD) indicating that cofilin-1 manipulation did not alter E-cadherin levels. Data is representative of three inserts from one donor. (M,N) Both knockdown and overexpression of cofilin-1 increased FITC–dextran permeability (M), and knockdown increased cellular velocity, while overexpression trended towards an increase without reaching statistical significance (N). Data is generated from four inserts from one donor. (O) Cofilin-1 knockdown increased the fraction of polymerized actin (representative blot, top panel and quantification, bottom panel) of cofilin-1 expression from air and CS-exposed NHBE transduced with adenovirus as indicated, as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of three inserts from one donor). (P) Cofilin-1 overexpression was sustained despite CS exposure (representative western blot, left panel and quantification, right panel) in NHBE transduced with adenovirus as indicated. Data is representative of four inserts from one donor. (Q,R) Cofilin-1 overexpression protected the monolayer from the CS-induced increase in (Q) FITC–dextran permeability, and (R) cellular velocity. Data is representative of three to four inserts from one donor. (S) Cofilin-1 over-expression protected from the CS-induced increase in MSD with time. (T) Representative images used for cell shape factor quantification in NHBE with knockdown or overexpression of cofilin-1 and in CS-exposed NHBE transduced with cofilin-1 overexpression. (U) NHBE with knockdown or overexpression of cofilin-1 demonstrated an increase in cell shape factor as compared to NHBE, whereas CS-exposed NHBE transduced with cofilin-1 overexpressing virus did not show statistical significant differences. The cell shape factor values were compared to NHBE values from Fig. 4L . Data is generated from three to four inserts from one donor. Graphs are generated with median bars. Statistics determined by Mann–Whitney test (A,C–H) or Kruskal–Wallis test followed by Dunn's multiple comparison test (J,K,M–R,U), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Binding Assay, Transduction, Knockdown, shRNA, Transfection, Virus, Over Expression, Fluorescence, Permeability, Generated, MANN-WHITNEY, Comparison

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: CS-exposed epithelia and COPD epithelia have cellular energetics that are distinct from pEMT. The COPD epithelia (CHBE) and CS-exposed NHBE were treated with LatA or U0126. The NHBE were treated with 2× EMT inducer supplement serve as a model for pEMT. (A) Ensembled average one-dimensional spatial energy spectra [E(κ), κ being wavenumber] of the horizontal velocity component is greatest for CS-exposed epithelium and CHBE, both which display classic slopes characteristic of Kolmogorov–Kraichnan turbulence as modeled by the −5/3 dashed line. Treatment of CS-exposed cells with the inhibitor of actin polymerization (LatA) or MAPK kinase inhibitor (U0126), decreased the energetics and turbulence in the monolayer approaching that of NHBE. (B) COPD epithelia (CHBE) also display classic slopes characteristic of Kolmogorov–Kraichnan turbulence, as modeled by the −5/3 dashed line. Treatment of CHBE with LatA or U0126 decreased the energetics and turbulence in the monolayer approaching that of NHBE. The pEMT cells have a completely different slope. (C) In monolayers with cofilin-1 knockdown (KD) or overexpression (OE) in NHBE, analysis was performed in the entire monolayer (brightfield image, BF) or in GFP–cofilin-1-manipulated cells only (GFP). The curve of cofilin-1 KD-specific cells (GFP) demonstrate a −5/3 slope similar to that of the CHBE cells, with similar energetics. The curve of the cofilin-1 OE-specific (GFP) cells have higher energetics, but similar slope to that seen in pEMT. (D) Biphasic relationship between cofilin-1 and cell velocity. There is a biphasic relationship of cell velocity as a function of cofilin-1 (NHBE exposed to air or cigarette smoke and treated with GFP control, KD and OE at 1×10 10 pfu/ml and CHBE donors).

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Knockdown, Over Expression, Control

Journal: Environmental Health Perspectives

Article Title: Seasonal Variations in Air Pollution Particle-Induced Inflammatory Mediator Release and Oxidative Stress

doi: 10.1289/ehp.7996

Figure Lengend Snippet: Production of ( A ) IL-6 by AM and ( B ) IL-8 by NHBE cells stimulated with coarse, fine, and ultrafine Chapel Hill pollution particles collected in four different months. The control IL-8 and IL-6 concentrations were 1.7 ± 0.2 ng/mL and 198 ± 5 pg/mL, respectively. Particles were added to NHBE cells at 11 μg/mL and to AMs at 50 μg/mL. *Tukey adjusted p -value < 0.05; n = 3–4 each. The dashed line denotes 1.0 (no change over control). The brackets indicate the groups that are significantly different by ANOVA and the Tukey subtest.

Article Snippet: Typically at confluency, NHBE cells in the 12-well plate (catalog no. 3512; Costar, Corning, NY) had a density of 300,000–500,000 cells/well in 2 mL of BEGM.

Techniques:

Journal: Environmental Health Perspectives

Article Title: Seasonal Variations in Air Pollution Particle-Induced Inflammatory Mediator Release and Oxidative Stress

doi: 10.1289/ehp.7996

Figure Lengend Snippet: Production of ROSs measured by DCF in ( A ) AM and ( B ) NHBE cells stimulated with coarse, fine, and ultrafine Chapel Hill pollution particles collected in four different months. Particles were added to NHBE cells at 11 μg/mL and to AMs at 50 μg/mL. In general, NHBE cells appeared to be more responsive to PM than the AMs because increases in DCF signals were similar between the two cell types even though NHBE cells were exposed to lower doses of PM. *Tukey adjusted p -value < 0.05; # p < 0.05 versus control (ratio = 1.0) by the one-group Student t -test; n = 3–4 each. The dashed line denotes 1.0 (no change over control). The brackets indicate groups that are significantly different by the Tukey subtest.

Article Snippet: Typically at confluency, NHBE cells in the 12-well plate (catalog no. 3512; Costar, Corning, NY) had a density of 300,000–500,000 cells/well in 2 mL of BEGM.

Techniques:

Journal: Environmental Health Perspectives

Article Title: Seasonal Variations in Air Pollution Particle-Induced Inflammatory Mediator Release and Oxidative Stress

doi: 10.1289/ehp.7996

Figure Lengend Snippet: Production of ROSs measured by DHR in ( A ) AM and ( B ) NHBE cells stimulated with coarse, fine, and ultrafine Chapel Hill pollution particles collected in four different months. Particles were added to NHBE cells at 11 μg/mL and to AMs at 50 μg/mL. In general, NHBE cells appeared more responsive to PM than were AMs because increases in DHR signals were similar between the two cell types even though NHBE cells were exposed to lower doses of PM. *Tukey adjusted p -value < 0.05; n = 3–4 each. The dashed line denotes 1.0 (no change over control). The brackets indicate groups that are significantly different by ANOVA and the Tukey subtest.

Article Snippet: Typically at confluency, NHBE cells in the 12-well plate (catalog no. 3512; Costar, Corning, NY) had a density of 300,000–500,000 cells/well in 2 mL of BEGM.

Techniques:

Journal: Environmental Health Perspectives

Article Title: Seasonal Variations in Air Pollution Particle-Induced Inflammatory Mediator Release and Oxidative Stress

doi: 10.1289/ehp.7996

Figure Lengend Snippet: Effects of PM from 12 different months on the release of ( A ) IL-6 in AMs and ( B ) IL-8 in NHBE cells. Particles were added to AMs at 50 μg/mL and to NHBE cells at 11 μg/mL. The data for December ultrafine PM were not available and were omitted in the figure; n = 3–4 each.

Article Snippet: Typically at confluency, NHBE cells in the 12-well plate (catalog no. 3512; Costar, Corning, NY) had a density of 300,000–500,000 cells/well in 2 mL of BEGM.

Techniques:

Journal: Environmental Health Perspectives

Article Title: Seasonal Variations in Air Pollution Particle-Induced Inflammatory Mediator Release and Oxidative Stress

doi: 10.1289/ehp.7996

Figure Lengend Snippet: Correlations between Cr and IL-8 release in NHBE cells incubated with ( A ) coarse, ( B ) fine, and ( C ) ultrafine PM from 12 months. The dashed line represents the linear regression line. The overall model p -values for coarse, fine, and ultrafine PM were 0.106, 0.003, and 0.036, respectively. R 2 = 0.6082 and 0.401 for fine and ultrafine PM, respectively.

Article Snippet: Typically at confluency, NHBE cells in the 12-well plate (catalog no. 3512; Costar, Corning, NY) had a density of 300,000–500,000 cells/well in 2 mL of BEGM.

Techniques: Incubation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: 15-LOX-1 and differentiation of primary NHBE cells. Primary NHBE cells were grown for 3 weeks in an undifferentiated state in immersion cultures or in air-liquid interface cultures to induce terminal differentiation into bronchial epithelial-like structures. Cells were then harvested and processed for quantitative real-time reverse transcription-polymerase chain reaction (panel A, differentiated vs. undifferentiated NHBE cells, P = 0.0005), Western blotting (panel B), and 13-HODE levels by liquid chromatography tandem mass spectroscopy (panel C, differentiated vs. undifferentiated NHBE cells, P = 0.0002). + represents 15-LOX-1 positive control (HCT-116 colon cancer cells transfected with 15-LOX-1 expression vector).

Article Snippet: We obtained from MatTek Corporation (Ashland, MA) primary NHBE cells (originally provided by Clonetics [San Diego, CA)]) grown in an in vitro system to form a pseudostratified, highly differentiated mucociliary epithelium that closely resembles the epithelial tissue of the respiratory tract (differentiated NHBE cells) ( Supplementary Fig. S1A ) and NHBE cells grown in an undifferentiated state in submerged (immersion) cultures (undifferentiated NHBE cells) ( Supplementary Fig. S1B ) ( 22 , 23 ).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Western Blot, Liquid Chromatography, Tandem Mass Spectroscopy, Positive Control, Transfection, Expressing, Plasmid Preparation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

doi: 10.1158/1940-6207.CAPR-10-0280

Figure Lengend Snippet: 15-LOX-1 and P16 mRNA and protein expression levels in cancer cell lines. A and B, A total of 128 cancer cell lines (Supplementary Table S1) were cultured and processed for 15-LOX-1 (panel A) and p16 (panel B) mRNA by quantitative real-time reverse transcription-polymerase chain reaction. Dots in the dot plots are means of triplicate measurements from each cell line. The relative expression levels were calculated relative to expression of the calibrator sample (differentiated NHBE cells). Solid lines represent the median value for each group. C, 15-LOX-1 relative expression levels in cancer cell lines compared to the level in Caco-2 cells terminally differentiated by sodium butyrate treatment. 15-LOX-1 mRNA measurements are as in panel A but with terminally differentiated Caco-2 cells as the calibrator sample. Dots in the dot plots are means of triplicate measurements from each cell line. The solid line represents the median value for the relative expression levels. D, 15-LOX-1 protein expression in cancer cell lines. Cell lines—including cell lines with 15-LOX-1 mRNA expression levels nearly equal to or greater than the level in differentiated Caco-2 cells or NHEK—were cultured, processed for Western blotting, and probed with 15-LOX-1 antibody. Three repeated experiments yielded similar results.

Article Snippet: We obtained from MatTek Corporation (Ashland, MA) primary NHBE cells (originally provided by Clonetics [San Diego, CA)]) grown in an in vitro system to form a pseudostratified, highly differentiated mucociliary epithelium that closely resembles the epithelial tissue of the respiratory tract (differentiated NHBE cells) ( Supplementary Fig. S1A ) and NHBE cells grown in an undifferentiated state in submerged (immersion) cultures (undifferentiated NHBE cells) ( Supplementary Fig. S1B ) ( 22 , 23 ).

Techniques: Expressing, Cell Culture, Reverse Transcription, Polymerase Chain Reaction, Western Blot