Journal: Scientific Reports

Article Title: The vomeronasal system of the newborn capybara: a morphological and immunohistochemical study

doi: 10.1038/s41598-020-69994-w

Figure Lengend Snippet: Antibodies and lectins used, with species of elaboration, dilution, manufacturer, and catalogue number.

Article Snippet: Later, (ii) the non-specific binding was blocked with 2.5% horse normal serum from the ImmPRESS reagent kit Anti-mouse IgG/Anti-rabbit IgG (Vector Laboratories, CA, USA) for 30 min. (iii) The primary antibody was then added at the corresponding dilution (Table ) and allowed to incubate overnight.

Techniques: Plasmid Preparation

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: Percentage of club cells expressing Creb1 is increased following repeated administration of IL-1B. (A) Representative images of dual label immunohistochemistry for Creb1 (brown) and clara cell secretory protein (red) in airway cross sections from Creb1 fl/fl Scgb1a1 cre or Creb1 fl/fl Scgb1a1 wt mice. Arrow indicates an example of CCSP-positive (club) cell expressing presumably nuclear staining for Creb1. Scale bar is 20 μm. (B) Percentage of club cells expressing Creb1. * = compared to vehicle control, p = 0.0013; ** = main genotype effect across treatments, p = 0.0055. (C) Density of club cells in airway. For B and C, individual points are data collected from a single mouse. n = 5 per group. Abbreviations: WT, wild type; Scgb1a1 cre , club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: IL-1B treatment increases mRNA abundance of secreted and tethered mucins. Abundance of mRNA in the murine lung for (A) Muc5ac, * = main effect of IL-1B treatment across genotype, p = 0.0246; (B) Muc5b, * = main effect of IL-1B treatment across genotype, p = 0.0342; (C) Muc1, * = main effect of IL-1B treatment across genotype, p = 0.0206; and (D) Muc4 * = main effect of IL-1B treatment across genotype, p = 0.0048. Bronchoalveolar lavage concentrations of (E) Muc5ac and (F) Muc5B. A trend for statistical significance for main genotype effect across treatment was observed for Muc5ac concentrations ( p = 0.054). For all panels, individual points are data collected from a single mouse: Creb1 fl/fl Scgb1a1 wt mice treated with vehicle ( n = 6) or IL-1B ( n = 7); Creb1 fl/fl Scgb1a1 cre mice treated with vehicle ( n = 8) or IL-1B ( n = 8). For panel E and F, a single mouse from the Creb1 fl/fl Scgb1a1 cre treated with vehicle group was a statistical outlier using Grubbs outlier test. Therefore, concentrations of Muc5ac and Muc5b from this mouse were not included. Abbreviations: WT, wild type; Scgb1a1 cre , club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques:

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: Conditional loss of club cell Creb1 modifies mucin secretion characteristics. (A) Abundance of mRNA in the murine lung for (A) Rab3D, * = main effect of genotype across treatment, p = 0.0280; (B) P2ry2, * = compared to vehicle control, p = 0.0233; Significant genotype x treatment interaction, p = 0.0142; (C) M3R, * = main effect of genotype across treatment, p = 0.0179; and (D) Muc4, * = main effect of IL-1B treatment across genotype, p = 0. 0048. (D) Density of goblet cells staining for Alcian-Blue/PAS under basal and methacholine stimulated conditions. Density is for cells with acidic mucins, as detected by dark blue staining. Main effect of methacholine, p < 0.0001. (E) Density of goblet cells staining for Alcian-Blue/PAS under basal and methacholine stimulated conditions. Density is for cells with neutral mucins, as detected by purple/magenta staining. Main effect of methacholine, p = 0.0036. Significant treatment x genotype interaction, p = 0.0069. * = compared to Creb1 fl/fl Scgb1a1 wt vehicle control under methacholine stimulated conditions, p = 0.0028; ** = to Creb1 fl/fl Scgb1a1 wt IL-1B under methacholine stimulated conditions, p = 0.0067. Post methacholine bronchoalveolar lavage concentrations of (F) Muc5ac and (G) Muc5b. (H) Mean immunohistochemistry signal intensity of Muc5b in the airway post methacholine stimulation. For all panels, individual points are data collected from a single mouse. For Panel F, one mouse from the Creb1 fl/fl Scgb1a1 cre + VEH group had Muc5ac concentrations below detection and was therefore not included in analysis. Abbreviations: WT, wild type; Scgb1a1 cre , club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle; MCh, methacholine.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: Conditional loss of club cell Creb1 diminishes IL-1B-mediated alterations in pulmonary mechanics. Basal airway resistance (A) , airway elastance (C) , tissue damping (E) and tissue elastance (G) as measured with a forced oscillation technique. Methacholine dose response curves normalized to basal values for airway resistance (B) , airway elastance (D) , tissue damping (F) and tissue elastance (H) . For Panels (A, C, E, G) , individual points are data collected from a single mouse. For Panel (B) , * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p = 0.0005. For Panel (D) , * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p < 0.0001. For Panel (F) , * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p < 0.0001. For Panel (H) , a significant genotype x treatment x methacholine dose interaction was noted, allowing for individual post hoc comparisons to be conducted. * = IL-1B treatment in wildtype mice compared to wild type vehicle controls; at 50 mg/mL, p = 0.0351; at 100 mg/mL, p < 0.0001. Legend key in Panel (B) applies to Panels (D, F, H) . n = 7 for Creb1 fl/fl Scgb1a1 wt mice treated with vehicle or IL-1B; n = 9 for Creb1 fl/fl Scgb1a1 cre mice treated with vehicle or IL-1B. Abbreviations: WT, wild type; Scgb1a1 cre , club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques:

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: Conditional loss of club cell Creb1 modulates inflammatory responses post methacholine. (A) Numbers of cells in the BAL. (B) Percentage of cells that were granulocytes in the BAL. * = main effect of genotype across treatments, p = 0.0418. (C) Expression of lung Il1r1 mRNA normalized to vehicle control wild type mice. * p = 0.0324. (D) . Heat map of genes that were differentially expressed in the murine lung due to treatment and/or genotype. Scale represents fold change. More details are available in . For Panels (A–C) , n = 7 for Creb1 fl/fl Scgb1a1 wt mice treated with vehicle or IL-1B; n = 9 for Creb1 fl/fl Scgb1a1 cre mice treated with vehicle or IL-1B. For Panel (D) , n = 6 mice per group. Abbreviations: WT, wild type; Scgb1a1 cre , club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle. Csf2 , granulocyte macrophage colony stimulating factor; Il10 , interleukin 10; Ly96 , myeloid differentiation factor 2; Mpo , myeloperoxidase; Stat4 , signal transducer and activator of transcription 4; Tlr8 , toll like receptor 8.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: CREB regulates transcripts necessary for goblet cell expansion in human cell line reportedly consisting of club cells. (A) Abundance of CREB1 mRNA in response to IL-1B and/or CREB inhibitor. * = compared to respective vehicle-treated cells, p < 0.05. (B) Abundance of BDNF mRNA; * = main effect of CREB inhibition across treatment, p = 0.0033. (C) Abundance of FOXA2 mRNA; * = main effect of CREB inhibition across treatment, p < 0.0001. (D) Abundance of SPDEF mRNA; * = main effect of CREB inhibition across treatment, p < 0.0001. (E) Abundance of FOXA2 mRNA in cells treated with recombinant CREB protein; * = compared to vehicle control, p = 0.0480. (F) Abundance of SPDEF mRNA in cells treated with recombinant CREB protein; * = compared to vehicle control, p = 0.0371. Abbreviations: IL-1B, Interleukin 1β; VEH, vehicle; CREB1 , cAMP responsive element binding protein 1; BDNF , brain derived neurotrophic factor; FOXA2 , forkhead box A2; SPDEF , SAM pointed domain containing ETS transcription factor.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques: Inhibition, Recombinant, Binding Assay, Derivative Assay

Journal: Frontiers in Physiology

Article Title: Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

doi: 10.3389/fphys.2023.1323865

Figure Lengend Snippet: IL-1B increases cAMP concentrations and CREB directly interacts with FOXA2. (A) Forskolin-treated NCI-H322 cells were subjected to ChIP assays using primers designed encompassing CRE sites (binding site for CREB). End-point PCR was used to assess template DNA amplification in CREB or IgG immunoprecipitated samples. Genomic DNA was used as positive control for the PCR reactions. (B) Abundance of IL1R1 mRNA in H322 cells treated with IL-1B and/or CREB inhibitor. * = main effect of IL-1B treatment across groups, p = 0.0035. (C) Concentrations of cyclic AMP (cAMP) in H322 cells treated with IL-1B for 8 h. * = compared to vehicle control, p < 0.0001. (D) Proposed mechanism of club cell CREB-dependent pathways contributing to mucin prosecretory effects of IL-1B. Dashed line represents hypothetical relationship that is supported by our data and the literature. IL-1B did not modify M3R and Rab3D, but loss of CREB did. Abbreviations: IL-1B, Interleukin 1β; VEH, vehicle; CREB1 , cAMP responsive element binding protein 1; FOXA2 , forkhead box A2; SPDEF , SAM pointed domain containing ETS transcription factor; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitated; NTC, no template control; gDNA, genomic DNA; Muc5ac , Mucin5ac; Muc5b , Mucin5b; Muc1 , Mucin1; Muc4 , Mucin4; Rab3D, RAS oncogene famil y; M3R , muscarinic receptor 3.

Article Snippet: Dual labeling for CCSP (rabbit anti-CCSP; 07-623, Millipore Sigma; 1:1000 dilution; 3 h), a marker of club cells, and Creb1 (mouse anti-Creb1; MA1-083, Invitrogen; 1:200 dilution; 3 h) was achieved using the ImmPRESS Duet Double Staining Polymer Kit (MP-7724, Vector Laboratories).

Techniques: Binding Assay, Amplification, Immunoprecipitation, Positive Control