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

Journal: bioRxiv

Article Title: Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline

doi: 10.1101/2021.07.29.454392

Figure Lengend Snippet: (A) Immunofluorescence (IF) for PNA-lectin (spermatid acrosomes; grey) and DAPI (nuclei; blue) in cross-sections of testes from G5 WT and G5 K82Δ mice in each stage including those that were not-stageable (N.S.) because they were devoid of spermatids, which are necessary for accurately staging tubules. Scale bar: 50 μm. (B and C) Quantitation of total tubules that are stageable or not-stageable in (B) G1 WT, G1 K82Δ mice, and acd/acd or (C) G5 WT, G5 K82Δ, and acd/acd mice. (D and E) Quantitation of the percentage of stageable seminiferous tubules in a given stage of spermatogenesis in (D) G1 WT and G1 K82Δ mice and (E) G5 WT and G5 K82Δ mice. (F) Quantitation of the breakdown of each stage within G1 WT, G1 K82Δ, G5 WT, G5 K82Δ, and acd/acd mice. White diagonal lines indicate % of tubules in stages I-III, black horizontal lines are stages IV-VI, grey vertical lines are stages VII-VIII, black diagonal lines are stages IX-XII, and black filled bars are not-stageable. (G) Quantitation of tubule phenotype denoted as either normal organization (white bar), disordered organization (grey bar), or empty tubules (black bar) in G1 WT, G1 K82Δ, G5 WT, G5 K82Δ, and acd/acd mice. (H and I) Quantitation of the percentage of seminiferous tubules with either normal, disordered, or empty tubule phenotype in (H) G1 WT, G1 K82Δ, and acd/acd mice and (I) G5 WT, G5 K82Δ, and acd/acd mice. acd/acd was used as a positive control of gonadal defect. In panels B, D, and H: G1 WT (grey filled circles), G1 K82Δ (grey open circles). In panels C, E and I: G5 WT (black filled squares), G5 K82Δ (black open squares). In panels B, C, H, and I: acd/acd (red open triangle).

Article Snippet: Antibodies and stains used are as follows: mouse anti-SCP3 (1:200; abcam, ab97672), rabbit anti-PLZF (1:200; Santa Cruz Biotechnology, sc-22839), rabbit-anti-Sox9 (1:666; Millipore/Sigma Aldrich, AB5535), goat anti-rabbit IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11008), goat anti-rabbit IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11011), goat anti-mouse IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11004), goat anti-mouse IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11001), Rat anti-BrdU (1:200; Oxford Biotechnology, OBT0030CX), rabbit anti-cleaved caspase-3 (1:400; Cell Signaling Technology, 9661S), Lectin PNA, Alexa Fluor 488 conjugate (1:1000; Invitrogen/Fisher Scientific; L21409), DAPI (1:1000; Kirkegaard & Perry Laboratories, 71-03-00).

Techniques: Immunofluorescence, Quantitation Assay, Positive Control

Journal: bioRxiv

Article Title: Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline

doi: 10.1101/2021.07.29.454392

Figure Lengend Snippet: (A, C, E) Immunofluorescence for (A) PLZF (undifferentiated spermatogonia; red) (C) SCP3 (spermatocytes; red), (E) SOX9 (Sertoli cells; red), DAPI (nuclei; blue), and PNA Lectin (spermatid acrosomes; grey) in cross-sections of testes from G5 WT, G5 K82Δ mice, and acd/acd mice. Scale bar: 50 μm. (B, D, F) Quantitation of the number of (B) spermatogonia, (D) spermatocytes, and (F) Sertoli cells per tubule in G1 WT, G1 K82Δ G5 WT, G5 K82Δ, and acd/acd mice. See methods for total number of mice and tubules analyzed.

Article Snippet: Antibodies and stains used are as follows: mouse anti-SCP3 (1:200; abcam, ab97672), rabbit anti-PLZF (1:200; Santa Cruz Biotechnology, sc-22839), rabbit-anti-Sox9 (1:666; Millipore/Sigma Aldrich, AB5535), goat anti-rabbit IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11008), goat anti-rabbit IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11011), goat anti-mouse IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11004), goat anti-mouse IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11001), Rat anti-BrdU (1:200; Oxford Biotechnology, OBT0030CX), rabbit anti-cleaved caspase-3 (1:400; Cell Signaling Technology, 9661S), Lectin PNA, Alexa Fluor 488 conjugate (1:1000; Invitrogen/Fisher Scientific; L21409), DAPI (1:1000; Kirkegaard & Perry Laboratories, 71-03-00).

Techniques: Immunofluorescence, Quantitation Assay

Journal: bioRxiv

Article Title: Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline

doi: 10.1101/2021.07.29.454392

Figure Lengend Snippet: (A-B) Immunofluorescence for (A) BrdU (proliferation; green) or (B) cleaved Caspase-3 (apoptosis; green); DAPI (nuclei; blue), and PNA Lectin (spermatid acrosomes; grey) in cross-sections of testes from G5 WT and G5 K82Δ mice. Scale bar: 50 μm. (C-D) Quantitation of the percentage of (C) BrdU-positive and (D) cleaved Caspase-3-positive cells per tubule in G1 WT, G1 K82Δ, G5 WT, and G5 K82Δ mice.

Article Snippet: Antibodies and stains used are as follows: mouse anti-SCP3 (1:200; abcam, ab97672), rabbit anti-PLZF (1:200; Santa Cruz Biotechnology, sc-22839), rabbit-anti-Sox9 (1:666; Millipore/Sigma Aldrich, AB5535), goat anti-rabbit IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11008), goat anti-rabbit IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11011), goat anti-mouse IgG, Alexa Fluor 568 (1:200; Invitrogen/ThermoFisher, A11004), goat anti-mouse IgG, Alexa Fluor 488 (1:200; Invitrogen/ThermoFisher, A11001), Rat anti-BrdU (1:200; Oxford Biotechnology, OBT0030CX), rabbit anti-cleaved caspase-3 (1:400; Cell Signaling Technology, 9661S), Lectin PNA, Alexa Fluor 488 conjugate (1:1000; Invitrogen/Fisher Scientific; L21409), DAPI (1:1000; Kirkegaard & Perry Laboratories, 71-03-00).

Techniques: Immunofluorescence, Quantitation Assay

Journal: BMC Microbiology

Article Title: Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

doi: 10.1186/s12866-015-0601-7

Figure Lengend Snippet: Identification of a UDP-GlcNAc transporter (TcNST1) of T. cruzi by in vivo complementation assays. K. lactis mutant (Kl3) cells were transfected with TcNST1, the K. lactis UDP-GlcNAc transporter (Kl UGT, positive control) or empty vector (pE4, negative control). Wild-type K. lactis cells were included in the analysis for comparison. Cells were grown as described in the section. After labeling with GS-II lectin (Alexa Fluor 488 conjugate), yeast cells were separated by flow cytometry in a FACS Canto II (Becton & Dickinson). a Representative histograms showing lectin binding transfected cells. b Mean fluorescence intensities of a representative experiment in triplicate. Error bars represent the standard deviations. * p < 0.01

Article Snippet: After labeling with GS-II lectin (Alexa Fluor 488 conjugate), yeast cells were separated by flow cytometry in a FACS Canto II (Becton & Dickinson). a Representative histograms showing lectin binding transfected cells. b Mean fluorescence intensities of a representative experiment in triplicate.

Techniques: In Vivo, Mutagenesis, Transfection, Positive Control, Plasmid Preparation, Negative Control, Comparison, Labeling, Flow Cytometry, Binding Assay, Fluorescence

Journal: EMBO Reports

Article Title: Cytoplasmic control of Rab family small GTP ases through BAG 6

doi: 10.15252/embr.201846794

Figure Lengend Snippet: A, B BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans‐ Golgi membrane protein Stx6 (green) in BAG6‐suppressed CHO cells with a Chinese hamster‐specific siRNA ( cBAG6 siRNA#2). Scale bar: 10 μm (A). Images of the cis‐ Golgi membrane protein GS28 and the cis‐ Golgi matrix protein GM130 in BAG6‐suppressed CHO cells with another Chinese hamster‐specific siRNA ( cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue). C Glycosylation of the IL‐2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag‐tagged WT IL‐2Rα protein was expressed in HeLa cells with (+) or without (−) BAG6 siRNA, and was immunoprecipitated with an anti‐Flag antibody. The precipitates were incubated with (+) or without (−) 10 unit of the deglycosylation enzyme PNGase F and subjected to Western blot analysis with an anti‐Flag antibody. Low‐mobility (indicated as glycosylated) and high‐mobility (indicated as non‐glycosylated) signals of WT IL‐2Rα are indicated. D–F Defects in the distribution of cell surface glycoproteins in BAG6‐suppressed cells. Representative image from a cell surface glycoprotein quantification assay with Alexa Fluor™ 488‐conjugated Lectin GS‐II as a probe (D). The graph quantitatively displays the number of fluorescence counts per cell as the mean ± SD calculated from 10 independent biological replicates (E). Lectin GS‐II‐derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment (see also Appendix Fig S6 ). * P < 0.05 compared with control knockdown (Student's t ‐test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down‐regulated the cell surface expression of glycoproteins (F). Source data are available online for this figure.

Article Snippet: Alexa Fluor™ 488 conjugate Lectin GS‐II was subsequently reacted at room temperature for 1 h or 4°C for 24 h, washing extensively with PBS, and cell surface fluorescent signals were detected by confocal microscopy LSM710 (Carl Zeiss).

Techniques: Confocal Microscopy, Staining, Immunoprecipitation, Incubation, Western Blot, Fluorescence, Derivative Assay, Software, Positive Control, Expressing