Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) CD11c + cells are increased in the lungs of atopic mice. Flow cytometry of lung cell suspension showing frequency (left) and cell number (right) at day 3 post inoculation (PI) high dose SeV ( B ) Adoptive transfer of CD11c + cells isolated from NA or atopic mice into naïve mice 24 h before inoculation with high dose SeV delays but does not prevent mortality; n=4 per group. ( C ) Transcriptomic (RNAseq) comparison between FACS isolated lung CD11c + cells from atopic and NA mice identifies several disparately expressed gene products, including Nrg1 (n=4 per group). Selected gene products shown. ( D ) NRG1 is markedly increased in atopic mouse lung (“tissue”), BAL, and supernatant from 1 x10 6 atopic lung CD11c + cells (“CD11c sup”) cultured for 24 h. ( E ) The scRNA (10x Platform) tSNE coordinates show CD14 + monocytes and dendritic cell (“DC”) sub-populations expressing NRG1 in peripheral blood cells of healthy human donors.*p<0.05, **p<0.01, ****p<0.0001

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Flow Cytometry, Suspension, Adoptive Transfer Assay, Isolation, Comparison, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) NRG1(1ng to 1000ng) i.n. (in 30µL) given daily to naïve mice for 5d before inoculation with high dose SeV reduces viral mortality; n=4 per group (1ng, 10ng 1000ng & PBS), n=8 per group (100 ng and 500 ng). ( B ) Ratio of EBD in the BAL to that in the lung shows reduced EBD in NRG1 treated mice on day 8 PI SeV. n≥8 per group, median ± IQR shown, Mann-Whitney U test.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) Adding NRG1 to hBEC inoculated with rgRSV (left) and mTEC inoculated with GFP-SeV (right) reduces spread of infection. Representative images shown. ( B ) Quantification of (A) for rgRSV and hBEC and ( C ) for GFP-SeV and mTEC. GFP positive cells quantified by ImageJ. Representative images from ≥3 separate experiments. *p<0.05, **p<0.01. ( D ) Transcriptomic analysis of hBEC cultures treated with NRG1 (100 ng) on the basolateral side of the Transwell for 5 days and inoculated with RSV (4000 pfu). RNA was isolated 48 h PI RSV and qRT-PCR performed using a custom Prime PCR array plate: (i) Transcripts in which NRG1 treatment reduced gene expression from that seen in RSV infected cells. (ii) Transcripts with low level expression that show small but significant change in expression relative to naïve control with NRG1 alone or genes significantly increased with RSV but whose expression levels were not affected by NRG1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Infection, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: Regionalized cell and gene signatures govern oesophageal epithelial homeostasis

doi: 10.1101/2024.02.21.581361

Figure Lengend Snippet: (A-B) Circular plot displaying number of interactions between proximal fibroblasts and proximal epithelial cells (A) and between distal fibroblasts and distal epithelial cells (B). Epithelial cells were aggregated into basal, suprabasal, and proliferative cells based on the annotation of subclustered epithelial cells. (C) Circular plot of differential interaction strengths in cell-cell communication between fibroblasts and epithelial cells comparing datasets containing proximal epithelial cells and fibroblasts and distal epithelial cells and fibroblasts, respectively. (D) Information flow (sum of communication probabilities among all pairs of cell groups) of all significantly altered signalling pathways comparing proximal and distal derived cells. (E, F) Heatmaps of selected incoming (blue) (E) and outgoing (green) (F) signalling pathways of regional oesophageal fibroblasts and epithelial cells. (G) Violin plots displaying regional gene expression of expressed WNT ligands in epithelial basal cells. (H) In situ hybridization of Axin2 (red) and Wnt5a (green) on cross-sections of the proximal and distal oesophagus. Sections are counterstained with E-CADHERIN (ECAD, white) and DAPI (blue). Scale bar = 20 µm. Dotted line marks the epithelial-stromal border. (I-J) Violin plots displaying regional gene expression of selected BMP ligand (I) and Igf1 (J) in fibroblasts. (K-L) Violin plot displaying regional gene expression of Igf1r (K) and Nrg1 (L) in basal and suprabasal epithelial cells.

Article Snippet: All supplemented media are based on ENR lo medium and contain either, 250ng/ml recombinant IGF1 (Peprotech, #100-11), 250ng/ml recombinant WNT5A (R&D systems, #645-WN-010), 100ng/ml BMP4 (R&D systems, #5020-BP-010), 5ng/ml IL-17A (R&D systems, #421-ML-025/CF), or 100ng/ml recombinant NRG1 (Biotechne, #9875-NR-050).

Techniques: Derivative Assay, Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: Regionalized cell and gene signatures govern oesophageal epithelial homeostasis

doi: 10.1101/2024.02.21.581361

Figure Lengend Snippet: (A) Information flow (sum of communication probabilities among pairs of cell groups) of selected signalling pathways comparing datasets encompassing all proximal and distal cells within the single-cell dataset. (B, D, F, H) Comparison of the outgoing and incoming interactions strengths between proximal-distal fibroblasts and proximal-distal epithelial cells, focusing on (B) WNT-, (D) BMP-, (F) IGF- and (H) NRG-signalling. (C) In situ hybridization for Wnt4 (red), enriched in the epithelium. (E) In situ hybridization for Bmp7 (red). White arrows mark Bmp7 -positive stromal fibroblasts in close contact with the epithelium. (G) In situ hybridization for Igf1 (red), enriched in the distal, compared to the proximal, stroma. (I) In situ hybridization for Nrg1 (red), expressed in the proximal, but not distal, epithelium. All in situ hybridizations (C, E, G, and I) are performed on cross-sections of the proximal and distal oesophagus and counterstained with E-CADHERIN (ECAD, white) and DAPI (blue). Dotted lines mark the epithelial-stromal border. Scale bar = 20µm.

Article Snippet: All supplemented media are based on ENR lo medium and contain either, 250ng/ml recombinant IGF1 (Peprotech, #100-11), 250ng/ml recombinant WNT5A (R&D systems, #645-WN-010), 100ng/ml BMP4 (R&D systems, #5020-BP-010), 5ng/ml IL-17A (R&D systems, #421-ML-025/CF), or 100ng/ml recombinant NRG1 (Biotechne, #9875-NR-050).

Techniques: Comparison, In Situ Hybridization, In Situ

Journal: bioRxiv

Article Title: Regionalized cell and gene signatures govern oesophageal epithelial homeostasis

doi: 10.1101/2024.02.21.581361

Figure Lengend Snippet: (A) Quantification of proximal and distal organoid forming efficiency (OFE) comparing control medium and medium containing 100ng/mL BMP4. (B) Quantification of organoid size in control medium and medium supplemented with 250ng/mL IGF1. (C-D) Quantification of organoid forming efficiency (C) and size (D) in control medium and medium supplemented with 100ng/mL recombinant NRG1. (E) Quantification of organoid forming efficiency in the presence of 250ng/mL WNT5A. All quantifications were performed at day 6 of culture. For size quantifications each dot presents average size of all organoids. n=3-5. (F) Graphical summary illustrating the regionalized oesophageal stromal and epithelial cell distributions as well as signalling gradients. (A-E) Two-sided ratio paired t-test. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: All supplemented media are based on ENR lo medium and contain either, 250ng/ml recombinant IGF1 (Peprotech, #100-11), 250ng/ml recombinant WNT5A (R&D systems, #645-WN-010), 100ng/ml BMP4 (R&D systems, #5020-BP-010), 5ng/ml IL-17A (R&D systems, #421-ML-025/CF), or 100ng/ml recombinant NRG1 (Biotechne, #9875-NR-050).

Techniques: Recombinant

Journal: Advanced Science

Article Title: Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation

doi: 10.1002/advs.202402607

Figure Lengend Snippet: Immobilized NRG1 sustains phosphorylation of downstream NRG1‐ERBB2/3 pathways A) mRNA and proteins levels of ERBB2 and ERBB3 receptors. B) Western blot demonstrating phosphorylation of Akt, Erk1/2, MAP38 and SAPK‐JNK for up to 12 h. C) Quantification of the relative phosphorylation of the indicated kinase normalized by the total amount of each protein. The housekeeping protein, GAPDH served as loading control. All values are mean ± S.D. Each experiment was repeated three times.

Article Snippet: Non‐tissue culture plates with a hydrophobic surface were incubated overnight at 4 °C with NRG1‐Fc fusion protein (Sino Biological, Beijing China, 100 µg mL −1 ) in phosphate‐buffered saline (PBS) containing 0.9 m m CaCl 2 and 0.9 m m MgCl 2 .

Techniques: Western Blot, Control

Journal: Advanced Science

Article Title: Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation

doi: 10.1002/advs.202402607

Figure Lengend Snippet: Differentiation on immobilized NRG1 upregulated mature SC markers at the mRNA level. A) PCA plot of the indicated RNA‐seq samples showing that KC‐NC that are differentiated in iNRG1 (SC_NRG‐Fc) are clustered separately from those differentiated with sNRG1 on collagen (SC_Col1) and naïve KC‐NC on day 7 or 14. B) Heatmap of notable SC genes among samples of Day 7 and 14 post KC‐NC induction and differentiation sNRG1 or iNRG1. C) Verification of RNA‐seq by qRT‐PCRs of immature (Pre‐SC), myelinating and de‐differentiating SC genes. D) Doubling times of naive KC‐NC and differentiated to SC on iNRG1.

Article Snippet: Non‐tissue culture plates with a hydrophobic surface were incubated overnight at 4 °C with NRG1‐Fc fusion protein (Sino Biological, Beijing China, 100 µg mL −1 ) in phosphate‐buffered saline (PBS) containing 0.9 m m CaCl 2 and 0.9 m m MgCl 2 .

Techniques: RNA Sequencing Assay

Journal: Advanced Science

Article Title: Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation

doi: 10.1002/advs.202402607

Figure Lengend Snippet: Immobilized NRG1 enhances Erk1/2 mediated YAP1 nuclear translocation and integrin expression. A) Western blot demonstrates sustained phosphorylation of Erk1/2 on iNRG1 as compared to soluble NRG1. B) qRT‐PCR for Hippo pathway effectors Lats1, Yap, Taz , and Tead1/2 . C) Immunostaining for YAP1 localization in KC‐NC, SC on Collagen 1 or iNRG1 and during Erk1/2 inhibition by LY3214996 and quantification of nuclear YAP. D) qRT‐PCR for integrin subunits α 6, α 7, and β 1 for the conditions mentioned above. Scale bars, 50 µm. All values are mean ± S.D. Each experiment was repeated three times.

Article Snippet: Non‐tissue culture plates with a hydrophobic surface were incubated overnight at 4 °C with NRG1‐Fc fusion protein (Sino Biological, Beijing China, 100 µg mL −1 ) in phosphate‐buffered saline (PBS) containing 0.9 m m CaCl 2 and 0.9 m m MgCl 2 .

Techniques: Translocation Assay, Expressing, Western Blot, Quantitative RT-PCR, Immunostaining, Inhibition