Journal: Cell Discovery

Article Title: RhoA-mediated G 12 -G 13 signaling maintains muscle stem cell quiescence and prevents stem cell loss

doi: 10.1038/s41421-024-00696-7

Figure Lengend Snippet: a RT-qPCR analysis of Ednrb , Ntsr1 , and Ntsr2 expression in freshly isolated MuSCs (FSC) and activated stem cells (ASC) ( n = 3). b Representative images of immunostaining for ET B /NTS 1 /NTS 2 (red), PAX7 (green) and DAPI (blue) on FDB myofibers of WT mice at 0 h (upper panel) and 24 h (lower panel) of culturing. c The quantification of GPCR staining intensity ( n = 3). d , e Quantification of the ratios of PAX7 + KI67 + ( d ) and PAX7 + MYOD ‒ ( e ) MuSCs on FDB myofibers after 24 h culturing in the presence of DMSO, BQ788 or SR142948A ( n = 6). f , g Quantification of the ratio of PAX7 + KI67 + ( f ) and PAX7 + MYOD − ( g ) MuSCs on FDB myofibers after 24 h culturing in the presence of DMSO, ET-3, NT, ET-3 + BQ788, NT + SR142948A, or ET-3 + NT ( n = 6). h ‒ l Representative images ( h ) and quantification of PAX7 + ( i ), the ratio of PAX7 + KI67 + ( j ), and PAX7 + MYOD + ( k ) cells, and quantification of MuSCs outside the basal limina ( l ) on transverse sections of WT mice TA muscle after intramuscular injection of solvent, BQ788 (1 mg/kg bodyweight), or SR142948A (0.5 mg/kg bodyweight), respectively ( n = 3). The cartoon in h depicts the outline of the experimental design. The data represent means ± SEM, analyzed by unpaired t -test ( a , c ) and one-way ANOVA with Bonferroni’s multiple comparisons test ( d ‒ g and i ‒ l ). ND not detected. Scale bars: 5 µm in b , 10 µm in h .

Article Snippet: The whole length cDNA of human EDNRB and NTSR2 genes were cloned from EDNRB-Tango (# 66458, Addgene) and NTSR2-Tango (# 66458, Addgene) plasmids, respectively, and inserted into the pcDNA3.1(+) vector subsequently.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Immunostaining, Staining, Injection, Solvent

Journal:

Article Title:

doi:

Figure Lengend Snippet: NLC drive expression of NTSR2-TrkB and pro-survival signaling in B-CLL cells. ( a ) Brightfield microscopy of co-cultured NLC (red arrows) and B-CLL cells (blue arrows). ( b ) Confocal microscopy analysis of CD14 (green) and CD68 (red) (left panel), and CD163 (green) and CD68 (red) (right panel), expression by NLC (n = 3). Nuclei are stained with DAPI. ( c ) Western blot analysis of CD68, CD163, and CD206 expression by NLC (n = 3). ( d ) Representative western blots showing expression of NTSR2, TrkB, p-Src, Src, and Bcl-2 by B-CLL cells isolated from two independent patients and cultured alone or with autologous NLC for 48 h. ( e ) Quantification of NTSR2 (n = 15), TrkB (n = 6), p-Src/Src (n = 17) and Bcl2 (n = 16) expression in independent patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05 and ** p < 0.01). Blots are cropped for clarity; full-length blots are shown in the Supplementary Fig. .

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Expressing, Microscopy, Cell Culture, Confocal Microscopy, Staining, Western Blot, Isolation

Journal:

Article Title:

doi:

Figure Lengend Snippet: The effects of BDNF on B-CLL pro-survival signaling are similar to those of BAFF, APRIL, and CXCL12 combined. ( a ) Representative western blot showing expression of NTSR2, p-Src, Src, and Bcl-2 by B-CLL cells isolated from patients. Cells were either co-cultured for 48 h with autologous NLC or cultured alone in complete medium supplemented (as indicated) with exogenous human (h) CXCL12 (100 ng/mL), BAFF (2 ng/mL), APRIL (25 ng/mL), or BDNF (100 ng/mL). (b,c,d), full-length blots are shown in the Supplementary Fig. . Quantification of NTSR2 ( b ), p-Src ( c ), and Bcl-2 ( d ) expression in four different patient samples. Data are presented as the mean ± SEM from at least three independent experiments, (* p < 0.05, ** p < 0.01). Blots are cropped for clarity; full blots are shown in the Supplementary Fig. .

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Isolation, Cell Culture

Journal:

Article Title:

doi:

Figure Lengend Snippet: Inhibiting BDNF in addition to BAFF, APRIL, and CXCR4 reverses NLC-mediated protection of B-CLL cells from apoptosis. ( a ) Representative western blot showing expression of p-Src and Bcl-2 by B-CLL cells isolated from patients. Cells were cultured (for 72 h) alone, with autologous NLC, or with autologous NLC plus single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). ( b – d ), full-length blots are shown in the Supplementary Fig. .Quantification of NTSR2 ( b ), p-Src ( c ) and Bcl-2 ( d ) expression in six independent experiments using six different patient samples. ( e ) Flow cytometry analysis of cell death, assessed by annexin V-fluorescein isothiocyanate/propidium iodide dual staining of B-CLL cells cultured (for 72 h) either alone, with autologous NLC, or with autologous NLC and single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 inhibition (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). Cell death was assessed by exclusion of annexin V/propidium iodide-negative cells. Experiments were performed using n = 9 different patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). ns not significant. ( f ) Schematic representation of the obtained results. NLC produce and secrete BDNF, which promotes survival of B-CLL cells by activating the Src signaling pathway and upregulating expression of Bcl-2. This newly described member of the NLC secretome appears to exert both complementary (alongside BAFF, APRIL and CXCL12) and independent effects. Here, we propose a model in which BDNF or pro-survival cytokines secreted by NLC within survival centers balance each other out to facilitate survival of B-CLL cells, and argue that simultaneous inhibition of BDNF signaling through the NTSR2-TrkB conditional oncogenic platform, along with inhibition of BAFF, APRIL and CXCR4/CXCL12, could cancel out NLC-mediated protection of B-CLL cells from apoptosis and restore normal cell death.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Isolation, Cell Culture, Inhibition, Flow Cytometry, Staining

Journal: Alcoholism, clinical and experimental research

Article Title: Heightened Exploratory Behavior Following Chronic Excessive Ethanol Drinking: Mediation by Neurotensin Receptor Type 2 in the Anterior Paraventricular Thalamus.

doi: 10.1111/acer.14406

Figure Lengend Snippet: Fig. 3. Chronic high ethanol (EtOH) drinkers exhibit reduced mRNA expression of neurotensin receptor type 2 (NTS2R) in the anterior paraventricular thalamus (aPVT), as assessed using quantitative real-time PCR (Experiment 3). (A) Comparison of the high EtOH drinkers (n = 6), low EtOH drinkers (n = 9), and water drinkers (controls) (n = 8) for mRNA levels of neurotensin (NTS), neurotensin receptor type 1 (NTS1R), and NTS2R in the aPVT fol- lowing 11 weeks of EtOH access (Week 12) during acute abstinence. Compared to low EtOH drinkers and water drinkers, the high EtOH drinkers showed significantly reduced expression of NTS2R mRNA in the aPVT. (B) Comparison of these same groups for the same measures in the posterior paraventricular thalamus revealed no group differences. Data are mean SEM, *p < 0.05.

Article Snippet: Tissue was immunotagged with polyclonal rabbit anti-NTS2R antibody (1:200, Cat # AP01326PU-N; Origene) and monoclonal mouse anti-NeuN antibody (1:1,000, Cat # ab104224; Abcam) and labeled with fluorescent goat anti-rabbit conjugated to AF488 (1:10,000, Cat # ab8245; Abcam) and goat anti-mouse conjugated to AF594 (1:500, Cat # A11005; Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: Alcoholism, clinical and experimental research

Article Title: Heightened Exploratory Behavior Following Chronic Excessive Ethanol Drinking: Mediation by Neurotensin Receptor Type 2 in the Anterior Paraventricular Thalamus.

doi: 10.1111/acer.14406

Figure Lengend Snippet: Fig. 4. Chronic high ethanol (EtOH) drinkers exhibit elevated protein levels of neurotensin receptor type 2 (NTS2R) in the anterior paraventricular tha- lamus (aPVT), as analyzed using Western blot (Experiment 4). (A) Comparison of the high EtOH drinkers (n = 6), low EtOH drinkers (n = 11), and water drinkers (controls) (n = 5) for protein levels of NTS2R in the aPVT following 11 weeks of EtOH access (Week 12) during acute abstinence. Compared to low EtOH drinkers and water drinkers, the high EtOH drinkers showed significantly elevated expression of NTS2R protein in the aPVT. (B) Comparison of these same groups for the same measures in the posterior paraventricular thalamus revealed no group differences. (C) Representative Western blot showing protein bands of NTS2R (green) and loading control GAPDH (red) in a water drinker (control), a low EtOH drinker, and a high EtOH drinker. (D) Original, full-size version of the Western blot, showing protein bands of NTS2R (green) and loading control GAPDH (red) in adult whole brain lysate for control (B), water drinkers (W), low EtOH drinkers (L), and high EtOH drinkers (H). Data are mean SEM, *p < 0.05.

Article Snippet: Tissue was immunotagged with polyclonal rabbit anti-NTS2R antibody (1:200, Cat # AP01326PU-N; Origene) and monoclonal mouse anti-NeuN antibody (1:1,000, Cat # ab104224; Abcam) and labeled with fluorescent goat anti-rabbit conjugated to AF488 (1:10,000, Cat # ab8245; Abcam) and goat anti-mouse conjugated to AF594 (1:500, Cat # A11005; Thermo Fisher Scientific).

Techniques: Western Blot, Comparison, Expressing, Control

Journal: Alcoholism, clinical and experimental research

Article Title: Heightened Exploratory Behavior Following Chronic Excessive Ethanol Drinking: Mediation by Neurotensin Receptor Type 2 in the Anterior Paraventricular Thalamus.

doi: 10.1111/acer.14406

Figure Lengend Snippet: Fig. 5. Elevated neurotensin receptor type 2 (NTS2R) signaling in the anterior paraventricular thalamus (aPVT) stimulates exploratory behavior (Experiment 5). (A) Average daily ethanol (EtOH) intake for low EtOH drinkers (n = 16) compared to high EtOH drinkers (n = 9) was significantly different at each week starting at Week 3. (B) Immunolabeling of NTS2R (green) and NeuN (neuronal nuclei; red), and nuclear staining by 40,6-diamidino-2- phenylindole (DAPI; blue) in the aPVT and posterior paraventricular thalamus (pPVT) shows expression of NTS2R in both neuronal cells (yellow) and nonneuronal cells (green). Images were taken at 10X magnification (left; scale bar: 100 µm) and 40X magnification (right; scale bar: 25 µm). (C) Sche- matic showing injection sites (black dots) for animals that received injections in the aPVT. Animals with a misplaced cannula, resulting in injections outside of the aPVT, were removed from analysis. Adapted from The Rat Brain, 5th edition, G. Paxinos and C. Watson, Copyright 2005, with permission from Elsevier. B = bregma. (D) Stimulating the NTS2R in the aPVT with the selective NTS2R agonist, JMV-431 (0.1 nmol or 1.0 nmol), compared to saline vehicle (0.3 µl), at Week 8 of EtOH access in low EtOH drinkers did not alter EtOH intake at any of the time periods measured during a 24-hour drinking session. (E) Stimulating the NTS2R in the aPVT with JMV-431 (1.0 nmol), compared to baseline or saline vehicle (0.3 µl), at Week 10 of EtOH access in low EtOH drinkers stimulated exploratory behavior in the light–dark box. Data are mean SEM, *p < 0.05.

Article Snippet: Tissue was immunotagged with polyclonal rabbit anti-NTS2R antibody (1:200, Cat # AP01326PU-N; Origene) and monoclonal mouse anti-NeuN antibody (1:1,000, Cat # ab104224; Abcam) and labeled with fluorescent goat anti-rabbit conjugated to AF488 (1:10,000, Cat # ab8245; Abcam) and goat anti-mouse conjugated to AF594 (1:500, Cat # A11005; Thermo Fisher Scientific).

Techniques: Immunolabeling, Staining, Expressing, Injection, Saline

Journal: BMC Medical Genomics

Article Title: Pro-neural transcription factors as cancer markers

doi: 10.1186/1755-8794-1-17

Figure Lengend Snippet: NTR2 expression increases progressively during the cancer development whereas NT and Ascl1 expression is upregulated at the BPH-PIN transition . (a) Immunohistochemical analysis of prostate tissue microarrays comprising normal, BPH, PIN and adenocarcinomas. Sections were stained with anti-NT, anti-NTR2 or anti-Ascl1 antibodies. NTR2 immunoreactivity is most intense in PIN and carcinomas, in contrast NT and Ascl1 immunoreactivity is mostly observed in the carcinomas. (b-d) Histopathological score for each specific prostate tissue based on the immunoreactivity antibody staining.

Article Snippet: For NT (Sigma), NTR2 (Acris Antibodies GmbH) and Ascl1 (Aviva Systems Biology) antibodies, antigen retrieval was performed in Tris-EDTA at pH 9.0 in a microwave for 15 minutes.

Techniques: Expressing, Immunohistochemical staining, Staining