Journal: Thoracic Cancer

Article Title: S‐ and R‐Carvedilol Prevent Benzo(a)pyrene‐Induced Lung Carcinogenesis

doi: 10.1111/1759-7714.70109

Figure Lengend Snippet: Effects of S‐ and R‐carvedilol on BPDE‐induced lung epithelial cell transformation: Benzo(a)pyrene diol epoxide (BPDE), an active metabolite of B(a)P, was used to transform BEAS‐2B cells. (A) & (B) The cells were pre‐treated with 5 μM S‐ and R‐carvedilol, 5 μM atenolol, 5 μM propranolol (Prop), and 5 μM ICI‐118551 (ICI) for 2 h, and then treated with 0.2 μM of BPDE for 1 h. The cells were then cultured with 5 μM of the drugs for 7 days. Afterward, they were seeded in soft agar in a 96‐well plate (2000 cells/well) containing 5 μM of the drugs in the top layer of agar. The cell colonies were counted under a microscope after 10 days of incubation. (C) Representative images were taken using GelCount of the wells containing colonies that grew on agar after 10 days of incubation. The plotted data are represented as mean ± SD, n = 6 to 8. The one‐way ANOVA followed by Dunnett's multiple comparison test was used to assess statistical differences. Statistical significance was denoted as ****: P < 0.0001 and **: P < 0.01.

Article Snippet: The BEAS‐2B cell line, originating from normal human bronchial epithelial cells, was obtained from ATCC (catalog number CRL‐9609).

Techniques: Transformation Assay, Cell Culture, Microscopy, Incubation, Comparison

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Schematic representation of workflow to identify CF correctors. Differentially expressed genes from CF patients (ΔF508-CFTR homozygous) were used for connectivity mapping to identify CF candidate therapeutics. These candidate therapeutics were further prioritized by incorporating additional layers of information from systems biology of CF. Prioritized CF candidate therapeutics were experimentally validated using intestinal organoids and bronchial epithelial cells from CF patients.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques:

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: FIS in ΔF508/ΔF508-cftr intestinal organoids. Representative images of intestinal spheres demonstrating fluid secretion in response to the test compounds (Panel A). Bar graph represents quantitation of fluid secretion in the intestinal organoids (Panel B). In this preliminary screening assay, all compounds were tested at a dose of 10 µM, while the known correctors (VX-809 and VX-661) were tested at a dose of 2 µM. The highlighted compounds (PP2 and LY294002) in the bar graph were found to show forskolin-induced swelling significantly better than that seen with the treatment of VX-809 or VX-661. The related raw, and processed data for FIS assay is made available as Additional file 4.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay, Screening Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: PP2 Dose response experiments in mice. Panel A. Representative images of ΔF508/ΔF508- cftr enterospheres depict secretion under various treatment conditions: (i) PP2 (0, 0.1, 1 and 10 µM, 24 h), (ii) PP2 (1 µM, 24 h) + CFTR inh -172 (20 µM, 30 min), (iii) VX-809 (2 µM, 24 h), and (iv) PP2 (1 µM, 24 h) + VX-809 (2 µM, 24 h). Panel B . Line graph represents quantitation of fluid secretion in enterospheres under various treatment conditions as described previously (Panel A).

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Preclinical validation of PP2. Panel A shows representative images of intestinal spheres demonstrating forskolin-induced swelling (FIS) in enteroids from normal subject and those from cystic fibrosis patients in response to DMSO, PP3, VX-809, and PP2. The bar graph represents quantitation of fluid secretion in the intestinal organoids. Panel B. Western blot data depict bands B (immature or endoplasmic reticulum form) and C (mature or membrane form) of CFTR immunoprecipitated from HEK 293 cells that overexpressed FLAG ΔF508-CFTR with and without treatment with PP2 (0.5 and 2 µM, 24 h), VX-809 (2 µM, 24 h) alone, and PP2 + VX-809 (2 µM each, 24 h). Panel C . Representative CFTR-mediated short-circuit currents ( I sc ) in human bronchial epithelial cells carrying ΔF508/ΔF50-CFTR in response to VX-809, PP2, and VX-809+PP2 treatment. Bar graphs represent data quantification of maximal increase in I sc /cm 2 from n = 5 (DMSO-treated) and n=6 (PP2-treated) independent traces. Panel D . Predicted binding pose of PP2 to ATP binding site.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay, Western Blot, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Functional enrichment network of differentially expressed genes following treatment with PP2 (2 µM; 48 h) in human CFBE (ΔF508/ΔF508- CFTR )

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Functional Assay

Journal: Journal of Innate Immunity

Article Title: Midkine Is Part of the Antibacterial Activity Released at the Surface of Differentiated Bronchial Epithelial Cells

doi: 10.1159/000346709

Figure Lengend Snippet: Production of MK by airway epithelial cells in vitro and contribution to bactericidal activity of the air surface liquid. a Primary HBEC were cultured in an ALI for 4 weeks to allow differentiation, in the absence of antibiotics. The medium was changed, the apical side rinsed with buffer, and the cells were incubated for another 24 h, whereafter the underlying medium was collected and the apical surface was rinsed with buffer. The amount of MK was determined by ELISA and the results shown represent the mean and SD from three separate experiments. b HAEpiC, primary HBEC, the bronchial epithelial cell lines BEAS-2B and 16-HBE, and the alveolar epithelial cell line A549 were grown to near confluence in a flask. The cell medium was changed and the cells incubated for 72 h, the medium collected and the MK content determined by ELISA. No MK could be detected in the medium from the A549 cell line (data not shown). The results represent the mean and SD from three different incubations. c, d To investigate possible bactericidal activity in the air surface liquid, HBEC were grown to confluence and allowed to differentiate using the air-liquid system in the presence of RA. After removal of mucus, the apical surface was rinsed with buffer which was incubated with S. pneumoniae (strain TIGR4). One part was used for the viable count assay and one part was processed for SEM. Bacteria incubated with the rinsing fluid show blebbing and disturbed integrity (d), compared with bacteria incubated in buffer alone (c). e To determine whether MK contributes to the bactericidal activity of the rinsing fluid, MK was immunoprecipitated from one portion of the rinsing fluid while another portion was immunoprecipitated using control antibodies. Thereafter, the rinsing fluids were used to investigate bactericidal activity against S. pneumoniae (strain TIGR4), using the viable count assay. The rinsing fluid depleted of MK showed significantly lower bactericidal activity compared with the rinsing fluid that had been immunoprecipitated with control antibodies, suggesting that MK constitutes a significant part of the bactericidal activity in airway surface liquid. Statistical significance was determined using Student's t test for paired observations. IP = Immunoprecipitation.

Article Snippet: Human pulmonary alveolar epithelial cells (HAEpiC) comprised of type 1 and type 2 pneumocytes (3H Biomedical) were grown in poly- L -lysin (Sigma) coated flasks in alveolar epithelial cell medium with supplements (3H Biomedical).

Techniques: In Vitro, Activity Assay, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

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: bioRxiv

Article Title: HDAC6 inhibitor ACY-1083 shows lung epithelial protective features in COPD

doi: 10.1101/2022.03.21.485098

Figure Lengend Snippet: Mucociliary transport rates were determined by tracking fluorescent microspheres placed on top of cell layer of unchallenged or TNF-challenged HBEC ALI cultures with or without ACY-1083 treatment. (A) Images of tracked beads in the ImageJ software with the Fiji plugin Tracking. Lines show movement of beads. (B) Microsphere speeds determined from three COPD donors and three fields per well. Box plot with dots representing three COPD donors and whiskers showing min and max. Data was normalized to the DMSO-control without TNF for each donor (set to 100%). Statistical analysis was performed using one-way ANOVA with Dunnett&s multiple comparisons test.

Article Snippet: Primary COPD human bronchial epithelial cells (HBEC) were purchased from Lonza (Basel, Switzerland).

Techniques: Software