Journal: Cell chemical biology

Article Title: A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75 NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

doi: 10.1016/j.chembiol.2018.09.007

Figure Lengend Snippet: Figure 1. Small-Molecule NSC49652 Affects the Interaction of p75NTR TMDs (A) Volcano plot of AraTM screening assay on NCI Diversity V compound library. A total of 1,580 compounds dispensed in twenty 96-well plates were screened at 50 mM in three independent runs. Fold change was calculated against the corresponding vehicle (DMSO) control of each plate. Hits were defined as compounds resulting in greater than ±0.5-fold change in GFP/optical density at 630 nm (OD630) signal without affecting OD630 by greater than 0.3-fold across the three runs. Compound NSC49652 is indicated. (B) Chemical structure of NSC49652. (C) Dose response of NSC49652 in the AraTM assay of p75NTR TMDs. Results are plotted as means ± SD (N = 3). The half maximal inhibitory concentration (10 mM) was calculated as the dose that resulted in half maximal response.

Article Snippet: Antibodies against p75NTR extracellular domain were obtained from Alomone Labs (ANT-007).

Techniques: Screening Assay, Drug discovery, Control, AraTM Assay, Concentration Assay

Journal: Cell chemical biology

Article Title: A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75 NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

doi: 10.1016/j.chembiol.2018.09.007

Figure Lengend Snippet: Figure 2. Solution Structure of the Complex between p75NTR TMD and NSC49652 and Structure-Function Analysis of TMD Resi- dues Engaged by NSC49652 (A) Superposition of backbone heavy atoms of the ten lowest-energy structures of the human p75NTR

Article Snippet: Antibodies against p75NTR extracellular domain were obtained from Alomone Labs (ANT-007).

Techniques:

Journal: Cell chemical biology

Article Title: A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75 NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

doi: 10.1016/j.chembiol.2018.09.007

Figure Lengend Snippet: Figure 3. NSC49652 Alters the Relative Conformation of p75NTR TMDs and Induces Dynamic Changes in the Full-Length Recep- tor in Mammalian Cells (A) Structural comparison of p75NTR TMD dimers in the presence (red) and absence (brown) of NSC49652 (green). The disulfide bond is indicated in yellow. The model without drug was derived from PDB: 2MIC (Nadezhdin et al., 2016), produced un- der the same conditions used in the present study. The two models are shown alone (left) and super- imposed to each other (right) to highlight their dif- ferences. Angle changes induced in the complex with NSC49652 are indicated. (B) Live cell homo-FRET anisotropy of p75NTR in COS cells in response to NSC49652. Shown are representative traces of average anisotropy change after addition of NSC49652 (20 mM at 0 min) or vehicle in cells expressing wild-type human p75NTR. (C) Live cell homo-FRET anisotropy of full-length rat p75NTR in COS cells in response to NGF. Shown are representative traces of average anisotropy change after addition of NGF (50 ng/mL at 0 min) or vehicle (PBS) in cells expressing wild- type human p75NTR. (D) Live cell homo-FRET anisotropy of wild-type and P254G mutant (rat numbering, equivalent to human Pro253) p75NTR in COS cells in response to NSC49652. Shown are representative traces of average anisotropy change after addition of NSC49652 (at 0 min). (E) Live cell homo-FRET anisotropy of wild-type and C257A mutant (rat numbering, equivalent to human Cys256) p75NTR in COS cells in response to NSC49652. Shown are representative traces of average anisotropy change after addition of NSC49652 (at 0 min).

Article Snippet: Antibodies against p75NTR extracellular domain were obtained from Alomone Labs (ANT-007).

Techniques: Comparison, Derivative Assay, Produced, Expressing, Mutagenesis

Journal: Cell chemical biology

Article Title: A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75 NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

doi: 10.1016/j.chembiol.2018.09.007

Figure Lengend Snippet: Figure 4. NSC49652 Induces Apoptosis through p75NTR and the JNK Pathway in Neurons and Affects Viability of Melanoma Cells (A) Representative western blot analysis showing induction of apoptotic markers (cleaved caspase-3 and PARP) by NGF and NSC49652 in cultures of post-natal day 5 (P5) rat cerebellar granule neurons (CGNs) assessed by western blotting. Blots were reprobed with antibodies against actin. The experiment was repeated three times with comparable results. (B) Representative western blot analysis showing induction of cleaved caspase-3 by NSC49652 in the presence and absence of JNK inhibitor JNK-IN-8 (3 mM) in cultures of P5 rat CGNs assessed by western blotting. Blots were reprobed with antibodies against actin. The experiment was repeated three times with comparable results. (C–E) Representative western blot analysis showing induction of cleaved caspase-3 (C and E) or PARP (D) by NSC49652 in cultures of P7 mouse CGNs derived from wild-type (WT) mice and p75NTR knockout (C), DDD (D), or C259A (E) mutant mice. Blots were reprobed with antibodies against p75NTR extracellular domain and actin. The experiment was repeated three times with comparable results. (F–G) Representative western blot analysis showing induction of cleaved PARP in human A875 (F), 70W, and 3S5 (G) cell lines in response to 4 hr treatment with the indicated concentrations of NSC49652. The experiment was repeated three times with comparable results. Unlike primary neurons, robust induction of cleaved caspase-3 was difficult to obtain in melanoma cells. (H) Representative western blot analysis showing induction of cleaved PARP in A875 control (NT [non-targeting]) and knockdown (shp75) cells in response to NSC49652 (20 mM) or anisomycin (3.8 mM). The experiment was repeated three times with comparable results. (I) Dose-dependent cell viability of A875 control (NT) and knockdown (shp75) cells in response to NSC49652. Results are plotted as means ± SD (N = 3). *p < 0.05; **p < 0.01.

Article Snippet: Antibodies against p75NTR extracellular domain were obtained from Alomone Labs (ANT-007).

Techniques: Western Blot, Derivative Assay, Knock-Out, Mutagenesis, Control, Knockdown

Journal: Cell chemical biology

Article Title: A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75 NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

doi: 10.1016/j.chembiol.2018.09.007

Figure Lengend Snippet: Figure 5. Oral Administration of NSC49652 Reduces Tumor Growth and Improves Survival in a Human Melanoma Xenograft Model (A) Quantitative assessment of tumor burden as measured every other day using a caliper. NSC49652 was administered orally during the first 3 weeks. Data are presented as means ± SEM (N = 10 mice per group). *p < 0.05; n.s., not significant. A875-GFPLuc NT (non-targeting), control cell line; A875-GFPLuc shp75, p75NTR knockdown cell line. (B) Kaplan-Meier assessment of survival following treatment with NSC49652 or vehicle as indicated. p < 0.0001 (log rank test) for NSC49652 compared with vehicle in A875-GFPLuc NT. (C) Representative images of formalin-fixed, paraffin-embedded tumor sections stained by TUNEL from vehicle- or NSC49652-treated animals bearing A875- GFPLuc NT tumors. Total cell nuclei were visualized using DAPI- (blue) and apoptotic TUNEL-positive cells (fluorescein-dUTP) are green. Scale bar, 15 mm. (D) Apoptotic index in vehicle- or NSC49652-treated A875-GFPLuc NT tumors expressed as the ratio of the number of TUNEL-positive cells to the number of DAPI-positive cells per field of view. Data are expressed as means ± SEM from three independent fields (two mice/group) using a 403 microscopic field. Means that are significantly different from controls are indicated as ***p < 0.001.

Article Snippet: Antibodies against p75NTR extracellular domain were obtained from Alomone Labs (ANT-007).

Techniques: Control, Knockdown, Staining, TUNEL Assay

Journal: Frontiers in Molecular Biosciences

Article Title: USF1 Transcriptionally Regulates UGT1A3 and Promotes Lung Adenocarcinoma Progression by Regulating Neurotrophin Signaling Pathway

doi: 10.3389/fmolb.2022.758968

Figure Lengend Snippet: Knockdown of USF1 inhibited cells proliferation, migration and invasion via deregulation of the neurotrophin signaling pathway. (A) The protein expression of p75NTR, RIPK2, IRAK1, IKKβ and USF1, detected by WB in H1299 and A549 cells; (B) The levels of IRAK1 was detected by WB in H1299 and A549 cells; (C,D) Cells migration was detected by wound healing assay in H1299 and A549 cells; (E,F) Cells migration and invasion were detected by transwell assay in H1299 and A549 cells; (The sh-USF1, cells were transfected with sh-USF1; sh-USF1+pcDNA3.1, cells were transfected with sh-USF1 and pcDNA3.1; sh-USF1+ pcDNA3.1- IRAK1; * p < .05 means statistically significant difference when compared with sh-USF1+ pcDNA3.1 group).

Article Snippet: After that, membranes were incubated overnight at 4°C with rabbit polyclonal antibody anti-USF1 (abs115735, 1:500 dilution, Absin, Shanghai, China), rabbit UGT1A3 polyclonal antibody (H00054659-A01, 1:500 dilution, Abnova, Wuhan, China), and rabbit P75 neurotrophic factor receptor (P75NTR) polyclonal antibody (TA328682, 1:200 dilution, OriGene Technologies, Rockville, United States), rabbit receptor interacting serine/threonine kinase 2 (RIPK2) polyclonal antibody (abs130017, 1:500 dilution, Absin, Shanghai, China), rabbit interleukin-1 receptor-associated kinase 1 (IRAK1) polyclonal antibody (AF7290, 1:500 dilution, Beyotime Biotechnology, Shanghai, China), rabbit ikappaB kinase β (IKKβ) polyclonal antibody (AF7200, 1:300 dilution, Beyotime Biotechnology, Shanghai, China), while the rabbit anti-GAPDH polyclonal antibody (1:500, Cell Signaling Technology, Inc.; Shangahi, China) was used as a control.

Techniques: Knockdown, Migration, Expressing, Wound Healing Assay, Transwell Assay, Transfection

Journal:

Article Title: Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

doi: 10.1242/dev.01429

Figure Lengend Snippet: Dhh expression by progenitor populations in the developing nerve. We isolated p75+P0 − NCSCs (A; 83% of these cells formed multilineage colonies, and 6% formed colonies that contained myofibroblasts and glia in a clonal analysis), p75+P0 + NCSCs (A; 74% of these cells formed multilineage colonies, and 12% formed myofibroblasts and glia), p75−P0 + myofibroblast progenitors (A; 97% of these cells formed myofibroblast-only colonies) and p75−P0 − myofibroblast progenitors (A; 99% of these cells formed myofibroblast-only colonies) from E14.5 rat sciatic nerve (Morrison et al., 1999). Only small numbers of restricted glial progenitors (that form glial-only colonies in culture) are present in the E14.5 sciatic nerve, but the frequency of these cells increases from E15.5 to E17.5, as NCSCs undergo glial restriction (Morrison et al., 1999). As negative controls, we also isolated p75+α4+ gut NCSCs (B; 76% of these cells formed multilineage colonies) and p75−α4− gut epithelial progenitors (B; none of these cells gave rise to colonies that contained neural cells, although they did give rise to fibroblasts in culture). By quantitative (real-time) RT-PCR, we found that nerve p75+P0 − NCSCs expressed the highest levels of Dhh, followed by slightly lower levels in p75+P0 + NCSCs (1.45-fold lower), gut epithelial cells (15-fold lower), gut NCSCs (16-fold lower), and nerve myofibroblasts (20 to 40-fold lower) (C). As no reporter gene expression was observed within the intestines of Dhh-Cre+loxpRosa+ mice, the low level of Dhh expression in nerve myofibroblasts appears to be inconsistent with reporter gene activation within these cells. D shows a representative experiment in which cDNA was random primed and normalized based on β-actin content, and then Dhh expression levels were compared between samples. We confirmed that each reaction product was homogeneous based on the melting curve, and that a single product was generated of the expected size (data not shown). Sequencing of the Dhh band confirmed that the correct sequence had been amplified.

Article Snippet: Briefly, dissociated cells from the sciatic nerve and gut were stained with antibodies against the neurotrophin receptor p75 (192Ig) and α 4 integrin (Becton-Dickinson, San Jose CA, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Gene Expression, Activation Assay, Random Primed, Generated, Sequencing, Amplification

Journal:

Article Title: Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

doi: 10.1242/dev.01429

Figure Lengend Snippet: Some myofibroblast progenitors are neural crest derived. Wnt1-Cre mice were mated with β-actin-flox/stop-β-gal conditional reporter mice. Sciatic nerves were dissected from the fetuses at E13.5, dissociated and cultured at clonal density. Some cells formed mixed colonies containing neural cells as well as myofibroblasts (A), whereas other colonies contained only neural cells (B), or only myofibroblasts (C). Neural cells were p75+ (not shown), had a characteristically spindly morphology, and grew in relatively dense colonies (B). Myofibroblasts were p75−SMA+, were characteristically large and flat, and grew in small, dispersed colonies (C,D). The cultures were stained with X-gal to identify neural crest-derived cells (blue product). Colonies from Cre−reporter+ littermate controls were never X-gal+ (A; Table 2), but neural colonies from Cre+reporter+ mice were usually X-gal+ (B; Table 2), as would be expected. Myofibroblast colonies from Cre+reporter+ mice were sometimes X-gal+, indicating neural crest derivation (C). Similar results were obtained using Wnt1-Cre+loxpRosa+ mice, in which some nerve cells formed β-gal+SMA+ myofibroblast colonies (D; see Table 2 for quantification).

Article Snippet: Briefly, dissociated cells from the sciatic nerve and gut were stained with antibodies against the neurotrophin receptor p75 (192Ig) and α 4 integrin (Becton-Dickinson, San Jose CA, USA).

Techniques: Derivative Assay, Cell Culture, Staining

Journal:

Article Title: Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

doi: 10.1242/dev.01429

Figure Lengend Snippet: NCSC-derived myofibroblasts express the endoneurial fibroblast marker Thy1, and the generation of these cells is enhanced by Bmp4, Nrg1 and Delta. (A,D) After 14 days in standard medium, nerve NCSCs give rise to myofibroblasts that are either SMA+Thy1− (purple arrows; 46±17% of cells with myofibroblast morphology), SMA+Thy1+ (white arrows; 44±15%), or SMA−Thy1+ (white arrowhead; 10±3%). Note that these colonies also contain large numbers of neurons and glia that are not visible in this field of view. After only 6 days in standard medium (B,D), NCSCs give rise to colonies containing mainly undifferentiated cells, as well as small numbers of myofibroblasts, which are mostly (92±6%) SMA+Thy1− (purple arrows). (C,D) In 6 day cultures supplemented with Delta, Nrg1 and Bmp4, nerve NCSCs give rise to larger numbers of myofibroblast cells, that include a higher frequency of SMA+Thy1+ cells (white arrows; 60±8%), and SMA−Thy1+ cells (white arrowhead; 28±7%) and a lower frequency of SMA+Thy1− cells (not shown; 12±4%). *P<0.05, relative to 6 day No Add (standard medium without Delta, Nrg1 or Bmp4 added);∂ indicates P<0.05, relative to 14 day No add (D). (E) Neural crest-derived myofibroblast progenitors can be isolated from acutely dissociated fetal rat sciatic nerve as p75−P0 + cells. (F) On average, 40±11% of these p75−P0 + cells express Thy1 in vivo. (G) 100% of the colonies that arose in culture from these p75−P0+Thy1+ cells were myofibroblast-only, and about 83% of these colonies consisted exclusively of SMA+Thy1− cells.

Article Snippet: Briefly, dissociated cells from the sciatic nerve and gut were stained with antibodies against the neurotrophin receptor p75 (192Ig) and α 4 integrin (Becton-Dickinson, San Jose CA, USA).

Techniques: Derivative Assay, Marker, Isolation, In Vivo

Journal: PLoS ONE

Article Title: Fibroblasts Isolated from Human Middle Turbinate Mucosa Cause Neural Progenitor Cells to Differentiate into Glial Lineage Cells

doi: 10.1371/journal.pone.0076926

Figure Lengend Snippet: Two different morphologies of OECs were found. Both types were both GFAP and p75 NTR positive labeled. One type (A–D) with filamentous GFAP labeling (B) and punctate p75 NTR labeling (C). Arrow head identifies one cell with single p75 NTR labeling. Cells labeled only with p75 NTR were not counted as OECs in this study. Another type (E, F) had intense p75 NTR labeling (red) and diffuse GFAP labeling (green). Scale bars: 50 µm.

Article Snippet: The following primary antibodies were used: mouse anti-human Thy1.1 (1∶1200, Serotec), rabbit anti-human basic fibroblast growth factor (FGF2, 1∶100, Santa Cruz Biotechnology), mouse anti-human Nestin (1∶200, Chemicon), mouse anti-human chondroitin sulfate proteoglycan (CSPG, 1∶100, Chemicon), mouse anti-human fibronectin (1∶100, Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP, 1∶250, DAKO), mouse anti-human low affinity neurotrophin receptor p75 (p75 NTR , 1∶750, USBiological), and mouse anti-β Tubulin III (anti-Tubulin, 1∶75, Sigma).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Fibroblasts Isolated from Human Middle Turbinate Mucosa Cause Neural Progenitor Cells to Differentiate into Glial Lineage Cells

doi: 10.1371/journal.pone.0076926

Figure Lengend Snippet: The percentage of p75 NTR and GFAP positive cells in the total cells (A) from middle turbinates of 7 patients. Representative photographs of middle turbinate tissue: patient 1 (B) and patient 6 (C). In C, bone was separated from lamina propria. Note that the size of samples from two patients was similar, but the yields of OECs were very different.

Article Snippet: The following primary antibodies were used: mouse anti-human Thy1.1 (1∶1200, Serotec), rabbit anti-human basic fibroblast growth factor (FGF2, 1∶100, Santa Cruz Biotechnology), mouse anti-human Nestin (1∶200, Chemicon), mouse anti-human chondroitin sulfate proteoglycan (CSPG, 1∶100, Chemicon), mouse anti-human fibronectin (1∶100, Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP, 1∶250, DAKO), mouse anti-human low affinity neurotrophin receptor p75 (p75 NTR , 1∶750, USBiological), and mouse anti-β Tubulin III (anti-Tubulin, 1∶75, Sigma).

Techniques: