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: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: TGF-β-induced, ShcA-dependent gene expression profiling in ErbB2-expressing breast cancer cells identifies Chrdl1. (A) Immunoblot analysis of NMuMG-ErbB2/rtTA cells following doxycycline treatment for the indicated times using antibodies against ShcA, ErbB2, and α-tubulin. (B) Schematic of experimental design. (C) Gene ontology analysis of differentially expressed genes. (D) Gene expression heat maps of the most differentially expressed genes in ErbB2-expressing breast cancer cells following TGF-β treatment (2.5 ng/ml) (3 or 24 h) in the context of high (−Dox) or low (+Dox) ShcA levels. A yellow to blue gradient represents low to high gene expression levels. The red box highlights Chrdl1 Agilent gene expression data. (E and F) TGF-β-induced, ShcA-dependent gene expression of BMP family members or BMP antagonists in ErbB2-expressing breast cancer cells. Gene expression heat maps of BMP genes (E) or BMP antagonist genes (F) in breast cancer cells following TGF-β treatment (3 or 24 h) in the context of high (−Dox) or low (+Dox) ShcA levels are shown. A yellow to blue gradient represents low to high gene expression levels. The red box highlights Chrdl1 Agilent gene expression data. (G) RT-qPCR analysis of Chrdl1 mRNA expression levels following TGF-β stimulation in the presence or absence of ShcA. *, P < 0.029. (H) Chrdl1 secreted protein expression levels were monitored by ELISA using conditioned medium harvested under the conditions described for panel G. Secreted Chrdl1 concentrations were normalized to protein concentrations measured in whole-cell protein lysates. *, P < 0.023.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Gene Expression, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 expression is elevated following TGF-β stimulation of HER2+ and basal breast cancer cells in the context of reduced ShcA levels. (A to C) Immunoblot analysis of ShcA expression was performed in NIC (A), MDA-MB-231 (B), and BT549 (C) breast cancer cells transfected with scrambled or ShcA-specific siRNAs. Cells were stimulated with TGF-β (+) or not stimulated (−) for 24 (NIC and MDA-MB-231) or 48 h (BT549). In all cases, α-tubulin was used as a loading control. (D to F) ELISAs were performed to detect secreted Chrdl1 levels in the conditioned media of NIC (D), MDA-MB-231 (E), and BT549 (F) breast cancer cells. Secreted Chrdl1 concentrations were normalized to protein concentrations from whole-cell protein lysates. *, P = 0.044; **, P = 0.015; ***, P = 0.023.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Recombinant Chrdl1 suppresses BMP7-induced Smad1/8 phosphorylation in mouse breast cancer cells and Smad1/9 phosphorylation in human breast cancer cells but fails to block TGF-β-induced signaling. (A to C) Immunoblot analyses using antibodies against phospho-Smad and total Smad1 in MDA-MB-231 (phospho-Smad1/9) (A), HCC1954 (phospho-Smad1/9) (B), and NMuMG-ErbB2 (phospho-Smad1/8) (C) breast cancer cells treated with BMP7 (60 min, 50 ng/ml) and increasing concentrations of rChrdl1 (60 min, 100 to 400 ng/ml). (D to F) The ratio of phosphorylated Smads to total Smad1 protein in MDA-MB-231 (D), HCC1954 (E), and NMuMG-ErbB2 (F) breast cancer cells was measured using the Odyssey infrared imaging system, and the averaged fold increases relative to untreated controls (−BMP7 and −rChrdl1) from three independent experiments are shown. (G to I) BT549 (G), HCC1954 (H), and NMuMG-ErbB2 (I) breast cancer cells were serum starved overnight and stimulated with TGF-β (2 ng/ml) (+) or not stimulated (−) in the presence (+) or absence (−) of recombinant Chrdl1 (100 ng/ml) for 30 min. Immunoblot analyses were performed with antibodies against phospho-Smad2 and total Smad2/3. In all cases, α-tubulin was used as a loading control.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Recombinant, Phospho-proteomics, Blocking Assay, Western Blot, Imaging, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Stable reduction of ShcA levels increases Chrdl1 expression within ErbB2-expressing mammary tumors in vivo. Representative images of ErbB2/ShcAhigh (A) and ErbB2/ShcAlow (B) mouse mammary tumors following immunohistochemical (IHC) staining with antibodies against ShcA or Chrdl1 are shown. Scale bar in ×4 panel, 100 μm (applies to all panels). Scale bar in ×20 inset, 20 μm (applies to all panels).

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Immunohistochemistry

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Conditioned medium containing Chrdl1 suppresses BMP4-induced Smad phosphorylation in breast cancer cells. (A) Immunoblot analysis with antibodies specific for phospho-Smad and total Smad1 in cells with high or low Chrdl1 levels following BMP4 treatment (30 min, 2.5 ng/ml). (B) ELISAs were performed on conditioned media from the indicated cell populations. Secreted Chrdl1 concentrations were normalized to protein concentrations from whole-cell protein lysates. (C and D) Immunoblot analysis with antibodies against ShcA was performed on NIC (C) and MDA-MB-231 (D) breast cancer cells that were transfected with scrambled control or ShcA-specific siRNAs and stimulated with TGF-β (2 ng/ml) or not stimulated for 24 h. Conditioned medium was collected and added to serum-starved NIC and MDA-MB-231 cells, which were left untreated or immediately stimulated with BMP4 (2.5 ng/ml) for 30 min. (E and F) NIC (E) or MDA-MB-231 (F) breast cancer cells were lysed and subjected to immunoblot analysis with antibodies specific for phospho-Smad1/5/8 (NIC), phospho-Smad1/5/9 (MDA-MB-231), or total Smad1. In all cases, α-tubulin was used as a loading control.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Recombinant Chrdl1 diminishes BMP4-induced Smad phosphorylation in breast cancer cells. (A) Immunoblot analyses using antibodies against phospho-Smad1/8 and total Smad1 in NMuMG-ErbB2 breast cancer cells treated with BMP4 (30 min, 2.5 ng/ml) and increasing concentration of rChrdl1 (30 min, 25 to 250 ng/ml). (B) The ratio of phosphorylated Smad1/8 to total Smad1 protein in NMuMG-ErbB2 cells was measured using the Odyssey infrared imaging system, and the averaged fold increases relative to untreated controls (−BMP4 and −rChrdl1) from three independent experiments are shown. (C to E) Immunoblot analyses using antibodies against phospho-Smad and total Smad1 in NMuMG-ErbB2 (phospho-Smad1/8) (C), BT549 (phospho-Smad1/9) (D), and HCC1954 (phospho-Smad1/9) (E) breast cancer cells treated with BMP4 (30 min, 2.5 ng/ml) and rChrdl1 (30 min, 100 ng/ml). The ratio of phosphorylated Smads to total Smad1 protein in NMuMG-ErbB2 (C), BT549 (D), and HCC1954 (E) cells was measured using the Odyssey infrared imaging system and the fold increases relative to untreated controls (−BMP4 and −rChrdl1) are indicated at the bottom of each panel.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Concentration Assay, Imaging

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 blocks BMP4-induced but not TGF-β-induced cell migration of NMuMG-ErbB2 cells and BT549 breast cancer cells. Analysis of cellular velocity as measured by manual tracking of NMuMG-ErbB2 and BT549 cells is shown. Cells were stimulated with BMP4 (2.5 ng/ml) (A and C) or TGF-β (2 ng/ml) (B) in the absence (left panels) or presence (right panels) of rChrdl1 (100 ng/ml). The graphs represent the mean cellular velocity from one representative experiment (four experiments for NMuMG-ErbB2 cells and three experiments for BT549 cells). The arrow in each panel indicates the time that ligand (BMP4 or TGF-β), in the presence or absence of rChrdl1, was added to the cultures. *, P < 0.001.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Migration

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Clinical correlations between Chrdl1 expression and overall survival of lung, ovarian, and gastric cancer patients. Kaplan-Meier survival curves comparing Chrdl1 expression with overall survival in lung cancer (n = 1,926) (A), ovarian cancer (n = 1,581) (B) and gastric cancer (n = 1,305) (C) patients are shown. P values were calculated using the log rank test between patients with high versus low Chrdl1 expression by using a median split.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 impairs BMP4-induced breast cancer invasion and gelatin degradation. (A) Boyden chamber invasion assays were performed with HCC1954 and BT549 cells. The data represent the average from three (HCC1954) or four (BT549) independent experiments performed in triplicate. Representative images from each breast cancer cell line, under each condition, are shown. *, P < 0.001. (B) NMuMG-ErbB2 and BT549 cells treated with or without BMP4, in the presence or absence of Chrdl1, for 24 h were cultured on glass coverslips coated with green fluorescent protein (GFP) fluorescent-gelatin matrix. Cells were fixed and images captured using confocal microscopy. Quantification of matrix degradation is illustrated in the graph, and representative images from each experimental condition are shown. The data represent two independent experiments (20 images were quantified for each condition in each independent experiment). Arrows indicate areas of gelatin degradation. NMuMG-ErbB2: *, P = 0.0164; **, P = 0.044. BT549: *, P = 0.0373; ** P = 0.027.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Cell Culture, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 impairs BMP4-induced MMP2 and MMP9 enzymatic activity. (A) NMuMG-ErbB2 and BT549 breast cancer cells were stimulated with BMP4 (2.5 ng/ml) or not stimulated for 24 and 48 h. MMP2, MMP9, and MMP14 mRNA levels were measured by RT-qPCR. The data represent the means from three independent experiments performed in triplicate. No statistically significant differences in MMP mRNA expression were observed following BMP4 stimulation. (B and C) Conditioned media (CM) from NMuMG-ErbB2 (B) and BT549 (C) breast cancer cells stimulated with BMP4 (2.5 ng/ml) or not stimulated, in the presence or absence of rChrdl1 (100 ng/ml), for 48 h were harvested and analyzed for MMP activity by gelatin zymography. The gels were photographed, and band intensities were digitally quantified. The data represent the mean band intensity for each condition from three (B) or four (C) independent experiments. (B, left) *, P < 0.0022; **, P < 0.024; (B, middle) *, P < 0.022; **, P < 0.029; (C, left) *, P < 0.045; (C, middle) *, P < 0.016; **, P < 0.045.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Zymography

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Correlations between Chrdl1 expression and clinical outcomes in breast cancer patients. (A) Chrdl1 mRNA expression is elevated in the basal and luminal A subtypes. The average Chrdl1 mRNA levels in all breast cancer subtypes was compared to the average Chrdl1 mRNA levels in each individual subtype (Lum A, luminal A; Lum B, luminal B). (B to D) Kaplan-Meier survival curves comparing Chrdl1 expression with overall survival (OS) (B), disease-free survival (DFS) (C), or distant metastasis-free survival (DMFS) (D) in breast cancer (n = 1,115 patients). P values were calculated using the log rank test between patients with high versus low Chrdl1 expression by using a median split.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing

Journal: Cancer cell

Article Title: Resistance to epigenetic-targeted therapy engenders tumor cell vulnerabilities associated with enhancer remodeling

doi: 10.1016/j.ccell.2018.11.005

Figure Lengend Snippet: A. Heatmap demonstrating the average expression in naive and resistant cells for all RTK/GF genes associated with 1–4 gained enhancers and log2(FC) expression > 1 in resistant vs. naive cells. B-C. Average log2 FPKM expression for ERBB4 (B) and NRG1 (C) across JQ1 naive and resistant samples. Error bars represent SD. D-E. H3K27Ac ChIP-sequencing tracks for ERBB4 (D) and NRG1 (E). Enhancers gained in resistance are underlined in red. F. Western blot of SK-N-BE(2)-C cells engineered to overexpress GFP or ERBB4 and stimulated with vehicle (Veh) or recombinant NRG1 for 6 hr. Western blots are probed for downstream effectors of PI3K signaling. G. Long-term viability assays in SK-N-BE(2)-C cells overexpressing the indicated proteins and treated with vehicle (DMSO) or 1 μM JQ1. Data are presented as percent viable cells relative to the DMSO arm for each condition. Shown are mean values of quadruplicate points ± SD. (ns = not significant, **** p value < 0.0001, un-paired two sample Student t-test with Welch correction). H. Representative images of data presented in (G). I. Western blot analysis of naive and JQ1 resistant SK-N-BE(2)-C cells probed for ALK, ERBB4, and NRG1. Cells were treated with vehicle (Veh) or JQ1 for 24 hr. J-K. Effects of lapatinib (J) and crizotinib (K) treatment on viability in naive and JQ1 resistant SK-N-BE(2)-C cells. L. Western blot analysis of naive and JQ1 resistant Kelly cells treated with vehicle (Veh) or JQ1 for 24 hr. M-N. Effects of lapatinib (M) and crizotinib (N) treatment on viability in naive and JQ1 resistant Kelly cells. See also Figure S5.

Article Snippet: For ERBB4/NRG1 rescue experiments, cells were treated with 100 ng/mL recombinant human NRG1 (R&D systems) twice per day.

Techniques: Expressing, ChIP-sequencing, Western Blot, Recombinant

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 binds directly to p75 NTR receptor. (A) Competition binding assays of [ 3 H]-DHEA in the presence of increasing concentrations of BNN27, using membranes isolated from HEK293 cells transfected with the cDNAs of full-length p75 NTR or ECD-truncated p75 NTR ΔECD receptors (Ki: mean ± SEM of five independent experiments). Right panel depicts efficacy of transfection, assessed by Western blots. (B) Immobilized BNN27 pulls down p75 NTR receptor. Covalently linked BNN27-7- O -(carboxymethyl) oxime (BNN27-7-CMO) to polyethylene glycol amino resin (NovaPEG amino resin) was incubated with recombinant p75 NTR proteins, then centrifuged. Precipitation experiments show Western blot analysis of pellets and supernatants with specific antibodies against p75 NTR proteins, as described in Section “Materials and Methods.” (C, i) STD-NMR revealed the interaction between BNN27 and p75 NTR , further amplified upon NGF additions. No interaction of BNN27 with NGF alone was observed as suggested by the null STD amplification. (ii,iii) 1 H STD-NMR and the corresponding STD-reference spectra. The binding of BNN27 at p75 NTR is evidenced by the presence of BNN27 methyls (18, 19) at the STD spectra; their absence in the case of NGF indicates the lack of BNN27-NGF interaction.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Binding Assay, Isolation, Transfection, Western Blot, Incubation, Recombinant, Amplification

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: Superimposition of representative MD simulations snapshots of BNN27 bound at p75 NTR /NGF 2:1 and at p75 NTR /mutated proNGF 2:2 complexes. Superimposed frames of MD simulations performed at the p75 NTR /NGF 1:2 and p75 NTR /mutated proNGF 2:2 complexes with BNN27. The steroid analog (in yellow) is spontaneously inserted at the CRD4 interfacial region of p75 NTR (in blue)/NGF (in pink). At the modified p75 NTR (in brown)/mutated proNGF (in green), BNN27 (in red) spontaneously penetrates at the interfacial region located at p75 NTR CRD1-CRD2 junction.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Modification

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 activates p75 NTR signaling. (A) BNN27 induced the release of RhoGDI from p75 NTR in mouse embryonic fibroblasts (MEF cells). MEF cells were transfected with the plasmid cDNA of p75 NTR and control plasmid (Mock). Transfectants were exposed for 30 min with BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml), or Estradiol (E2) (100 nM), and lysates were immunoprecipitated with p75 NTR -specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75 NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Results are representative of three independent experiments. (B) Regulation of RhoA activity by BNN27 in MEF cells. Constitutively active RhoA protein (provided by the kit manufacturer) was used as positive control. Results are expressed as the mean of triplicate measurements ± SEM normalized to control. ∗ P < 0.05 versus control. (C) BNN27 induced the release of RhoGDI from p75 NTR in a dose-dependent manner. MEF cells were transfected with the plasmid cDNA of p75 NTR and control plasmid (Mock). Transfectants were exposed for 30 min with various concentrations of BNN27 (1, 10, or 100 nM), or NGF (100 ng/ml), and lysates were immunoprecipitated with p75 NTR -specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75 NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Results are representative of three independent experiments. (D) BNN27 induced the association of p75 NTR with its effectors RIP2 in MEF cells. MEF cells were transfected with the plasmid cDNA of p75 NTR . Transfectants were exposed for 30 min to vector (Control), BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml) or Estradiol (E2) (100 nM), and lysates were immunoprecipitated with p75 NTR -specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75 NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments. (E) Structure-function relationships in the interaction between BNN27 p75 NTR. MEF cells were transfected with the plasmid cDNAs of p75 NTR wt or p75 NTR mutants that lack the entire Extracellular Domain (ΔECD) or p75C257A and interactor RIP2. Transfectants were exposed for 30 min to vector (Control), BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml) or E2 (100 nM), and lysates were immunoprecipitated with p75 NTR -specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75 NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Activity Assay, Positive Control

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 protects against apoptosis primary mouse cerebellar granule neurons. (A) Neurotrophin receptors expression in cerebellar granule neuron cultures. Cerebellar granule neurons at 1 DIV express the pan-neurotropic receptor p75 NTR and TrkB, the selective receptor for BDNF, but they do not express TrkA. HEK293 cells transfected with the appropriate cDNA of neurotrophin receptors were used as positive controls. (B) Cell death in response to BNN27 treatment was assessed in p75 NTR wt (up-right) and p75 NTR knockout (KO) (down-right) cerebellar granule neurons (CGNs), identified by b-III tubulin immunostaining (green), using the TUNEL method (red). White arrows indicate TUNEL positive cells that do not express β-III tubulin (red nucleus) and thus represent a non- specific cell population whereas yellow arrows represent TUNEL and β-III tubulin double-positive cells (yellowish nucleus), which is the cell population that was count. Scale bar: 50 μm. (C) Quantification of TUNEL positive neurons CGN cultures (p75 NTR wt, left and p75 NTR KO, right) after 16 h serum withdrawal, incubation with BNN27, NGF or BDNF. Results are expressed as the mean ± SEM. (corrected to control) of three independent experiments, each performed in triplicate. ∗ P < 0.05 versus control (Student’s t -test).

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Expressing, Transfection, Knock-Out, Immunostaining, TUNEL Assay, Incubation

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 decreases cell death signaling in primary mouse cerebellar granule neurons. (A) Co-immunoprecipitation of RIP2 with p75 NTR in CGNs isolated from p75 NTR wt mice. Incubation of CGNs for 20 min with BNN27, DHEA and NGF induce the recruitment of RIP2 from p75 NTR . Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments. (B) Co-immunoprecipitation of RhoGDI with p75 NTR in CGNs isolated from p75 NTR wt mice. Incubation of CGNs for 30 min with BNN27, DHEA and NGF induce the release of RhoGDI from p75 NTR . Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Similar results were obtained in three independent experiments. (C) Levels of phosphorylated c-jun kinase (p-JNK) in CGNs isolated from wt and p75 NTR KO mice. BNN27, DHEA or NGF Fold change was calculated by densitometric scanning of phospho-JNK signals normalized to total JNK levels. Results are representative of three experiments. (D) Activated Caspase-3 in CGNs, isolated from wild-type (WT) and p75 NTR knockout (KO) mice. After 16 h serum withdrawal treatment. Fold change was calculated by densitometric scanning of cleaved-Caspase-3 signals normalized to actin levels.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Immunoprecipitation, Isolation, Incubation, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: Schematic illustration of the pro-survival effects of BNN27 in a p75 NTR -mediated manner in primary mouse cerebellar granule neurons. BNN27 activates p75 NTR receptors, leading to the control of pro-survival RhoGDI and RIP2 effectors while in parallel attenuating the activation of pro-apoptotic JNK and Caspase-3 factors.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Activation Assay