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: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Regeneration of epithelium was assessed by BrdU staining. (a) Representative image at 10X (scale bar of 50 µm) of Cdh1 fl/fl mice instilled with adeno-Cre recombinase (Ad5CMVCre-eGFP) to knockdown E-cadherin for 3 months shows decreases in BrdU as compared to Cdh1 fl/fl instilled with adeno-Ctrl (Ad-5CMVeGFP). Decreases in the fluorescence intensity of b E-cadherin and c BrdU in Cdh1 fl/fl mice instilled with adeno-Cre as compared to Cdh1 fl/fl mice instilled with adeno-Ctrl. Data is generated from eight mice. To knock down E-cadherin in the AT2 cells of mice lungs, Cdh1 fl/fl -Spc Cre mice were fed tamoxifen (TAM) for 30 days. These were compared to Cdh1 fl/fl -Spc Cre mice receiving a normal chow diet (ND). In vivo knockdown of E-cadherin in Cdh1 fl/fl -Spc Cre mice show decreases in BrdU expression as observed in the d representative images at 10X (scale bar of 25 µm), with decreases in the intensity of e E-cadherin and f BrdU in AT2 cells. Data is generated from five mice. Undifferentiated normal basal epithelial cells were transfected with Ad-GFP-U6-h-CDH1-shRNA to knock down E-cadherin, and undifferentiated COPD cells were transfected with Ad-GFP-U6-h-CDH1 to overexpress E-cadherin. We compared to respective undifferentiated Normal/COPD with Ad-GFP. g Representative image at 40× (scale bar of 25 µm) and h quantification showing decreased BrdU intensity in the normal epithelium with E-cadherin knockdown (Normal + shCDH1) and COPD at baseline (COPD + GFP) as compared to Normal + GFP. Also, COPD with overexpressed E-cadherin (COPD + CDH1) demonstrates increased BrdU intensity compared to COPD + GFP. Data are expressed as median bars and generated from three inserts from two donors.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: BrdU Staining, Knockdown, Fluorescence, Generated, In Vivo, Expressing, Transfection, shRNA

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Immunofluorescence at 40× (scale bar of 50 µm) of COPD human bronchial epithelial cells differentiated at week one to three of air–liquid interface (ALI) show decreased expression of E-cadherin and β-tubulin (ciliated cells marker) expression, increase expression of MUC5AC (goblet cell marker), without any changes in Cytokeratin 14 (Basal cell marker), as compared to non-diseased human bronchial epithelial (normal) cells.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: Immunofluorescence, Expressing, Marker

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: COPD cells at 4 to 6 weeks ALI were transfected Ad-GFP-U6-h-CDH1 (CDH1) to overexpress E-cadherin or Ad-GFP (GFP) as control at 2 × 10 9 pfu. COPD cells transfected with Ad-CDH1 result in a increased epithelial resistance, b reduced cellular velocity of COPD cells, and c increased mRNA expression of CDH1 . Overexpression of E-cadherin in COPD cells d – g increased mRNA expression of claudins— CLDN1 , CLDN3 , CLDN7 , and CLDN8 , h decreased mRNA expression of CLDN10, i increased mRNA expression of occludin (OCLN), j increased mRNA expression of tight junction protein 1 (TJP1), and k TJP2 was not altered. Data is expressed as median bars and generated from 4 to 12 transwells per condition from two donors. To induce the over-expression of E-cadherin in Cdh1 knock-in mice, mice tracheal epithelial cells (mTECs) were transfected with adeno-Cre (Ad5CMVCre-eGFP) at 2 × 10 9 pfu and were exposed to CS for 10 days. Overexpression of E-cadherin in mTECs protects against CS-induced epithelial functional phenotypes by l decreasing monolayer permeability, m decreasing the cellular velocity, and n protecting Cdh1 mRNA downregulation due to CS exposure. Data for l – m involves three to six transwells per condition. COPD cells treated with CDDO-Me restore epithelial function by o improving the epithelial resistance, p decreasing the cellular velocity, and q increasing the CDH1 mRNA expression of COPD cells as compared to age and gender-matched non-diseased epithelium (normal controls). Similarly, cigarette smoke (CS) exposed to healthy normal cells treated with CDDO-Me r restores epithelial resistance, s decreases cellular velocity, and t protects against CDH1 mRNA downregulation due to CS exposure. (Data for o – t is generated from 3 to 6 transwells from 2 donors). For the panels the same data for normal are used for a – k , and o – t in the figure. Data are expressed as median bars. Kruskal-Wallis test, followed by Dunn’s multiple comparison test was performed . P < 0.05 were considered statistically significant.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: Transfection, Control, Expressing, Over Expression, Generated, Knock-In, Functional Assay, Permeability, Comparison