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: Toxicology letters

Article Title: Mitochondrial dysfunction is associated with Miro1 reduction in lung epithelial cells by cigarette smoke

doi: 10.1016/j.toxlet.2019.09.022

Figure Lengend Snippet: CSE reduces mitochondrial function as measured by oxygen consumption rate (OCR) an indicator of oxidative phosphorylation (OXPHOS) in human lung epithelial cells. BEAS2B, NHBE and DHBE cells were treated with CSE (0.5–1.0%) for 1–2 hrs or 24 h. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was evaluated by using a Seahorse XFp analyzer in (A–B) BEAS2B, (C–D) NHBE and DHBE cells. CS-induced suppression of basal OCR, ECAR, maximal respiration, proton leak, ATP production and spare capacity. Data are mean ± SEM, n = 2–3 per group. *P < 0.05, **P < 0.01, vs respective controls.

Article Snippet: Cell culture and treatments Human small airway epithelial cells (SAEC), normal human bronchial epithelial cells (NHBE) from healthy non-smoking subjects and diseased human bronchial epithelial cells (DHBE) from patients with COPD were obtained from Lonza (Walkersville, MD), and grown as per the recommendations of the supplier in appropriate primary cell culture growth media.

Techniques: Phospho-proteomics

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