Journal: PLoS ONE

Article Title: Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

doi: 10.1371/journal.pone.0100633

Figure Lengend Snippet: 10 −7 M estradiol (black bars) increased FUT-4, -5 and -6 mRNA expression compared with vehicle control (white bars). Data are expressed as A) absolute change in C t value normalized to HPRT1 and B) fold increase in mRNA expression over vehicle control normalized using HPRT1. Values shown are mean ± SEM of 4 different donors performed in 3 biological replicates. Total fucose sugar residues in the cytoplasmic protein fractions of ALI cultures were determined by lectin binding assays. Fucose residues were significantly increased with 10 −7 M estradiol using fucose binding lectins, C-D) AAA, E-F) LTA and G-H) UEA-1. D, F, and H are densitometric quantifications of C, E, and G, respectively. The intensity of all bands in each lane in detecting total fucose residues were quantified using the software program Image J and normalized to total protein loaded per lane in milligrams. Data is expressed as fold increase over vehicle control. Values shown are mean ± SEM of experiments performed with N = 4 donors. * P<0.05, ** P<0.01 compared against vehicle control. Non-parametric t-tests were used in all statistical analyses.

Article Snippet: Membranes were incubated with primary antibodies against ER-α, ER-β, NFATc1, MUC5AC, or (LTA-lotus tetragonolobus asparagus pea: H1601-1, AAA-anguilla anguilla lectin from fresh water eel: H4901-1, and UEA-1-ulex europaeus lectin from gorse: H2201-1) lectins (EY Laboratories Inc., San Mateo, CA) followed by incubation of secondary HRP-conjugated anti-rabbit, anti-mouse and beta-actin antibodies for 1 h at room temperature, and visualized using an enhanced chemiluminescence substrate (Thermo Scientific, Ontario, CAN) and a Chemigenius imaging system (Syngene, Cambridge, UK).

Techniques: Expressing, Control, Binding Assay, Software

Journal: bioRxiv

Article Title: Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon

doi: 10.1101/2024.07.03.601781

Figure Lengend Snippet: A) Ex vivo mucus growth in adult GF and ConvR mouse colon after stimulation with bacterial MAMPs. B) Ex vivo mucus growth dose-response to P3CSK4 in adult GF and ConvR mouse colon. C) Ex vivo mucus growth in adult GF and ConvR mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors. D) AB/PAS stained tissue sections from ex vivo experiments illustrated in (A). Emptied upper crypt GCs (red arrows) and lower crypt cavitation (yellow arrows) indicated. E) Whole mount confocal imaging of adult GF mouse colon treated with fluorescent Dextran tracer. Images show x/y-axis (upper panel) and x/z-axis (lower panel) cross-sections illustrating Dextran uptake by an upper crypt GC (purple arrow). F) Ex vivo mucus growth in neonatal (P3, 5, 15) and post weaning (P33) rat colon stimulated with P3CSK4 in the presence or absence of Dynasore inhibitor. G) Standardized expression of genes (columns) encoding known and predicted secreted proteins upregulated in mucus from P9-adult compared to P1-P7 rats (see ) in GC subpopulations (rows) identified by scRNA-seq. “Secretion” row indicates evidence of secretion determined by prior annotation or in silico prediction of classical or non-classical secretion by SecretomeP. H) Quantification of the frequency of Tgm3 expressing GCs as a proportion of the total GC population in neonatal (P3, P9, P14, P19) and post-weaning (P24) colonic tissue sections from ConvR mice. I) Confocal micrographs of representative tissue sections from P3 and P14 ConvR mice stained for Tgm3 (green) or the GC-binding lectins WGA (grey) and UEA1 (red). An individual GC from each image is indicated (yellow dashed line). Data represents n=3-5 (A-F, H-I)) animals per group, as indicated. All data is pooled from at least 2 independent litters or experiments. All histograms show median and interquartile range. Statistical comparisons between groups by 2-way ANOVA and Fishers LSD (A, C, F) or Kruskall Wallis and uncorrected Dunn’s test (H); p<0.05 (*), <0.01 (**) <0.001 (***), <0.0001 (****). Image scale bars are 50µm (D, I) or 20µm (E). # note: ConvR data displayed in A and B is reproduced from our previous publication and is shown for illustrative purposes only.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/mL; Merck) in some cases supplemented with combinations of UEA1 Atto 488-conjugated lectin (10 µg/mL; Merck), UEA1 DyLight647-conjugated lectin (10 µg/mL; Vectorlabs) or WGA Alexa 555-conjugated lectin (10 µg/mL; ThermoFisher) for 15 min.

Techniques: Ex Vivo, Activation Assay, Staining, Imaging, Expressing, In Silico, Binding Assay

Journal: bioRxiv

Article Title: Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon

doi: 10.1101/2024.07.03.601781

Figure Lengend Snippet: A) Schematic of the senGC activation pathway highlighting known (black) and putative (red) pathway genes. B) Expression of known and putative senGC genes in FACS isolated colonic GCs and colonocytes determined by DESeq2 analysis of bulk RNA sequencing data. C) Comparison of gene expression ratios between P22 ConvR:GF mice and adult 3-week ConvD:GF mice quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly up (red) or down (blue) regulated by microbiota exposure in both P22 and ConvD mice are indicated. D) Proportion of unique and shared genes significantly regulated by microbiota exposure in P22 ConvR and adult 3-week ConvD mice, based on data shown in (C). E) Comparison of microbiota-dependent expression of known and putative senGC activation pathway genes (A-B) in P22 ConvR and adult 3-week ConvD mice. Subset of data shown in (C). Genes not significantly regulated by microbiota in either group (grey), or genes regulated in either P22 ConvR (purple), adult ConvD (yellow) or both groups (teal) are indicated. F) Relative expression (compared to GF) of Duox2 (left) and Nox1 (right) genes in ConvD (brown) and B. fragilis monoassociated (blue) mice from 1-4 weeks colonization. Expression determined by qRT-PCR of colonic RNA, normalized to Gapdh and Rplp0 expression. G) Expression of Duox2 (left) and Nox1 (right) genes in postnatal ConvR (purple; P3-33) and GF (teal; P9-P33) determined by DESeq2 analysis of bulk colonic RNA sequencing data. H) Confocal micrographs of fixed colonic tissue sections from ConvR WT mice stained for Duox2 (left) and Nox1 (right) mRNA by in situ RNA hybridization and counterstained by Epcam (grey). Duox2 or Nox1 expressing crypt regions indicated (yellow arrows). I) Confocal micrographs showing upper crypt GCs in fixed colonic tissue sections from ConvR, GF and ConvD mice stained for Duox2 (red), mucus (UEA1; green), Actin (grey) and DNA (blue). Duox2 or Nox1 expressing crypt regions indicated (yellow arrows). J) Ex vivo mucus growth in Duox2 fl/fl and Duox2 ΔIEC colon tissue treated with carbachol (CCh), LPS or P3CSK4. K) Ex vivo mucus growth in WT colon tissue treated with P3CSK4 in the presence or absence of the Nox1 inhibitor ML171. Data represents n=2-5 animals per group, as indicated. All data is pooled from at least 2 independent experiments or litters. All histograms show median and interquartile range. Statistical comparisons between groups by DESeq2 (B, C, E), Kruskal Wallis and Dunn’s multiple comparison (F) or 2-way ANOVA and Fishers LSD (J, K); p<0.05 (*), <0.01 (**) <0.001 (***), <0.0001 (****). Scale bars are 50µm (H) or 10µm (I).

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/mL; Merck) in some cases supplemented with combinations of UEA1 Atto 488-conjugated lectin (10 µg/mL; Merck), UEA1 DyLight647-conjugated lectin (10 µg/mL; Vectorlabs) or WGA Alexa 555-conjugated lectin (10 µg/mL; ThermoFisher) for 15 min.

Techniques: Activation Assay, Expressing, Isolation, RNA Sequencing Assay, Comparison, Quantitative RT-PCR, Staining, In Situ, Hybridization, Ex Vivo