Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: (A) Secreted FGFs and (B) IL-8 were measured by Luminex (A to I) or ELISA (J), respectively, in basal supernatants of primary human BAECs grown at air-liquid interface (BAEC-ALI) after exposure to F.p. for 24 hours. Each line represents a different donor. * denotes P < 0.05 using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test; unless the control group was zero, # denotes P < 0.05 using Wilcoxon paired t tests. (C) Secreted FGF1, FGF2, FGF4, FGF18, (D) IL-8, and (E) G-CSF were measured in supernatants from F.p.-stimulated precision-cut lung slices. Three precision-cut lung slices were incubated per well per donor and exposed to F.p. (10−7 M) for 24 hours, with three replicates per experiment. One of three experiments is shown. *P < 0.05 with a paired t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Luminex, Enzyme-linked Immunosorbent Assay, Control, Incubation

Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: (A) Normal human lung fibroblasts were cultured in 3D collagen-rich rafts, as described in Methods. Cells from five different donors were stimulated with a cocktail of FGF1, FGF2, FGF4, and FGF18, all at 10 ng/ml, for 24 hours before RNA isolation for RNA-seq. Differentially expressed genes (DEGs) where P < 0.05 are displayed. nRPKM, normalized reads per kilobase of transcript per million mapped reads. (B) Expression, in RPKM, of select DEGs is shown. *P < 0.05 by a paired t test. (C) The experimental setup depicts BAEC-ALI stimulation with F.p. (10−7 M) or diluent control for 24 hours and transfer of basal supernatant, mixed 50:50 with fresh medium, to fibroblast cultures for 24 hours. (D and E) Expression of CSF3 (D) and HAS2 (E) was measured in fibroblasts after exposure to supernatant from BAEC-ALI, as described above. Media, 100% fresh medium; Cont. Sup., 50% medium from diluent-exposed BAEC-ALI with 50% fresh medium; F.p. Sup., 50% medium from F.p.-exposed (10−7 M) BAEC-ALI with 50% fresh medium. Erdafitinib (Erd; 100 nM) was added, as indicated, to the fibroblast cultures. Data are expressed as fold change (FC) from fibroblasts cultured with medium alone. (F to H) F.p.-exposed (10−7 M) precision-cut lung slices were incubated in the presence (100 nM) or absence (diluent control) of erdafitinib. Three precision-cut lung slices were incubated per well for 24 hours, with three technical replicates per experiment. One of three experiments is shown. IL-8 (F), G-CSF (G), and hyaluronan (H) concentrations were measured by Luminex or ELISA. Error bars show SEM. *P < 0.05 with an unpaired t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Cell Culture, Isolation, RNA Sequencing, Expressing, Control, Incubation, Luminex, Enzyme-linked Immunosorbent Assay

Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: C57BL/6 mice were treated with 10 μg of HDM in 25 μl by the intratracheal route on days 0, 2, and 4. On days 14, 16, 18, 28, and 30, mice were given 10 μg of HDM + F.p. (1 mg/kg) with erdafitinib (30 mg/kg) or diluent control (−, HP-β-CD), as indicated. (A) Total and differential cell analysis of airway infiltrates in BAL. (B) BAL cytokines and hyaluronan were measured by Luminex and ELISA, respectively. Error bars show SEM. *P < 0.05 with Mann-Whitney test. (C and D) Lung inflammation severity (C) and airway epithelial goblet cell hyperplasia (GCH) severity (D) scores were determined by an anatomic pathologist, in a blinded manner, from hematoxylin and eosin– and Alcian blue/periodic acid–Schiff–stained tissue sections, respectively. Scale bars, 100 μm. Severity scores were compared by Kruskal-Wallis with Dunn’s multiple comparisons test. (E) Serum cytokines were measured by Luminex. A minimum of four mice was used per group, with one of two experiments shown. Error bars show SEM. *P < 0.05 with Mann-Whitney test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Control, Cell Analysis, Luminex, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: The signalling receptor MCAM coordinates apical-basal polarity and planar cell polarity during morphogenesis

doi: 10.1038/ncomms15279

Figure Lengend Snippet: ( a ) Upper panel, cartoon of MCAM-BD 1–3. Lower panel, yeast zygotes obtained after mating the bait strain containing pGBKT7-MCAM with the library strain containing pGADT7-FGF2, FGF4, and FGF8. BD, DNA-binding domain; AD, activation domain; +, positive control with p53-BD; and −, negative control with empty AD vector and BD-lambda. ( b ) Co-immunoprecipitation of MCAM/FGF4 and FGFR1/FGF4 with the protein lysate treated with or without the heparinase I and heparinase III (0.06 IU ml −l ). ( c ) Kinetic dissociation constant ( K D ) of FGF4/MCAM, FGF4/FGFR1, or FGF4/ FGFR2 complexes was measured using a surface plasmon resonance method. ( d ) Distribution of polarized MCAM and unpolarized FGFR1 on chemotaxing cells. The source concentration of FGF4 in the chemotaxis assay is 10 ng ml −l . ( e ) Co-localization of endogenous MCAM and the apical marker aPKCζ in chemotaxing cells. Scale bar, 20 μm. ( f , g ) Time-lapse live-cells imaging of endogenous MCAM ( f ) or exogenous MCAM-RFP ( g ) at the leading edge of chemotaxing cells.

Article Snippet: Anti-FGF3 (1:2,000, ab10830), anti-FGF8 (1:2,000, ab181492), anti-CDC42 (1:500, ab187643) and anti-PKCζ (1:2,000, ab119291) antibodies were from Abcam.

Techniques: Binding Assay, Activation Assay, Positive Control, Negative Control, Plasmid Preparation, Immunoprecipitation, SPR Assay, Concentration Assay, Chemotaxis Assay, Marker, Imaging