Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: Primers for qRT-PCR associating serotonin signaling pathway.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques:

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The relative expression of the targeted genes.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Expressing

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The relative protein expression.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Expressing

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The average percentage of TPH-1, SERT, 5-HTR2A, ERK1/2 and pERK1/2 positive pneumocytes, PAMs and bronchial epithelial cells in the control, degenerative mitral valve disease (DMVD) and degenerative mitral valve disease with pulmonary hypertension (DMVD with PH) groups.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Control

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The cytoplasmic expression of TPH-1, SERT and 5-HTR2A in pneumocytes of alveoli PAMs (top row) and bronchial epithelial cells (bottom row) of dogs in the control ( a , d , g , j , m , p ), degenerative mitral valve disease (DMVD) ( b , e , h , k , n , q ) and degenerative mitral valve disease with pulmonary hypertension (DMVD with PH) groups ( c , f , i , l , o , r ) presented as brown color (Labeled streptavidin-biotin, Immunohistochemistry, Mayer’s Hematoxylin counterstained, 400× magnification).

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Expressing, Control, Labeling, Immunohistochemistry

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The average percentage of TPH-1, SERT, 5-HTR2A, ERK1/2 and pERK1/2 positive pneumocytes ( a ), PAMs ( b ) and bronchial epithelial cells ( c ) in the control, degenerative mitral valve disease (DMVD) and degenerative mitral valve disease with pulmonary hypertension (DMVD with PH) groups. Data are presented as mean and standard deviation (bars). a Indicate significant difference at p < 0.05 compared to the control group. b,c Indicate significant difference at p < 0.05 between the DMVD and DMVD with PH groups.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Control, Standard Deviation

Journal: Veterinary Sciences

Article Title: Investigation of Genes and Proteins Expression Associating Serotonin Signaling Pathway in Lung and Pulmonary Artery Tissues of Dogs with Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease: The Preliminary Study

doi: 10.3390/vetsci9100530

Figure Lengend Snippet: The significant Spearman correlation between the relative expression of the targeted genes and proteins and the echocardiographic parameters.

Article Snippet: The membranes were incubated overnight at 4 °C with 1:500 mouse anti-TPH-1 antibody (mouse monoclonal anti-tryptophan hydroxylase antibody, Sigma-Aldrich, Darmstadt, Germany), 1:1000 mouse anti-SERT antibody (mouse monoclonal anti-SERT antibody, Advanced Target Solutions, Kentwood, MI, USA), 1:1000 mouse anti-5-HTR2A antibody (mouse monoclonal anti-SR-2A antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 rabbit anti-ERK1/2 antibody, 1:1000 rabbit anti-pERK1/2 antibody (rabbit monoclonal anti- Phospho Plus p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody Duet, Cell Signaling Technology, Danvers, MA, USA), and 1:1000 mouse anti-α-tubulin antibody (mouse monoclonal anti-alpha tubulin antibody, InvitrogenTM, Waltham, MA, USA).

Techniques: Expressing

Journal:

Article Title:

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Figure Lengend Snippet: 5-HT1b and 5-HT2a receptors are expressed in growth cones and filopodia. a–j Sensory neuron growth cones immunostained for serotonin receptors, 5-HT1b (green in a, red in g), 5-HT2a (green, d and h) as well as filamentous actin to highlight the extent of the growth cone (Phalloidin, red; b and e). The receptors 5-HT1b and 5HT2a were observed as a widespread punctate distribution throughout the growth cone (g–h). Punctate expression was evident in filopodia (j) with isolated 5-HT1b (green, *), 5-HT2a (red, #) as well as colocalised (arrowheads) puncta on filopodial shafts. There was prominent colocalization of 5-HT2a and 5-HT1b receptors in peripheral areas (i, arrow). Scale bars are 5 μm (a–i) and 1 μm (j). k Turning responses to serotonin were sensitive to chlorpromazine. Attractive turning of growth cones to 5HT-Lo (n = 7, p = <0.0001) and repulsion to 5HT-Hi (n = 8, p = 0.003) were significantly different when cultures were treated with chlorpromazine. l Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). (Mann–Whitney u-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5HT-Lo elicits growth cone attraction through the 5-HT2a receptor. a Growth cone attraction was measured in the presence of 5HT-Lo gradients (p = <0.0001) and TCB-2 gradients (p = 0.0002). Growth cone attraction to 5HT-Lo was abolished by pharmacological application of the selective 5-HT2a receptor antagonist, ritanserin (n = 17, p = 0.0001) and the PLC inhibitor, U-73122 (n = 9, p = 0.0018). b Growth cone attraction to 5HT-Lo was not perturbed when growth cones treated with a specific 5-HT1b antagonist, GR55562 (n = 8, p = <0.0001), or the 5-HT3 antagonist ondansetron (n = 8, p = 0.0082). Depletion of ER calcium with thapsigargin (n = 19, p = 0.0003) abolished attractive turning to 5HT-Lo, while inhibition of VGCCs with nifedipine (n = 15, p = 0.0330) had no significant effect. Turning angles were compared to vehicle and 5HT-Lo and only significant differences shown in (a) and (b). Kruskal–Wallis, Dunn’s multiple comparison test. c, d Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). Kruskal–Wallis, Dunn’s multiple comparison test

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Comparison

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5HT-Hi promotes growth cone repulsion through the 5-HT1b receptor. a Growth cone repulsion was measured in the presence of 5HT-Hi gradients (p = <0.0001) and CP-94253 gradients (p = 0.0018). Growth cone repulsion to 5HT-Hi was altered to levels indistinguishable from random growth (vehicle) with application of the selective antagonist to 5-HT1b, GR55562 (n = 16, p = <0.0001), by activating adenylate cyclase with forskolin (n = 8, p = 0.0003) and restoration of cAMP signals with Sp-cAMPs (n = 8, p = 0.0001). b Repulsion to 5HT-Hi was not perturbed when growth cones were treated with the 5-HT2a receptor antagonist ritanserin (n = 8, p = 0.002) or the 5-HT3 antagonist ondansetron (n = 10, p = 0.0003). Inhibition of ER calcium release with thapsigargin (n = 10, p = <0.0001) or calcium influx with nifedipine (n = 8, p = <0.0001) did not alter growth cone repulsion from 5HT-Hi. Turning angles were compared to vehicle and 5HT-Hi. c, d Axon extension was not perturbed by any pharmacological application (ns, p > 0.05). Turning angles were compared to vehicle and 5HT-Hi. (Kruskal–Wallis, Dunn’s multiple comparison test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Hi-C, Comparison

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of 5-HT receptors to the turning or motile side of growth cones. Schematic a showing growth cone divided into “near” and “far” regions with respect to micropipet position (located on the upper left side). b–d Growth cones immunostained after turning in response to 5HT-Lo and stained for 5-HT2a (green, b), 5-HT1b (red, c) and merged in (d). Dotted line separates the near and far regions of growth cone (near vs far). (e) Representative image of filopodia with 5-HT2a (green), 5-HT1b (red) receptor puncta. (f) There was no significant near/far bias in the number of filopodia following exposure to 5HT-Lo (n = 8, p = 0.9004), 5HT-Hi (n = 13, p = 0.5450) or BDNF (n = 10, p = 0.7907) compared to vehicle. (Data not normally distributed: Kruskal–Wallis, Dunn’s multiple comparison test). g There were significantly more (n = 36, p = 0.0033) 5-HT2a puncta in filopodia on the near side of growth cones exposed to 5HT-Lo compared to all other treatments. h There was no bias in 5-HT1b puncta in filopodia of growth cones. (Mann–Whitney U-test). (i-j) When entire growth cones were analysed, i 5HT-2a (n = 9, p = 0.0006) and j 5HT-1b (n = 9, p = 0.0028) receptor translocation was significantly biased in growth cones exposed to 5HT-Lo. All ratios were compared to vehicle (Data normally distributed: Shapiro–Wilk. One-way ANOVA, Tukey’s multiple comparison test). Scale bars are 5 μm (Fig. 7b–d) and 1 μm (Fig. 7e)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Staining, Comparison, MANN-WHITNEY, Translocation Assay

Journal:

Article Title:

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Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of membrane 5-HT2a receptors to the motile side of growth cones. a–h Representative growth cones exposed to vehicle and 5HT-Lo gradients (micropipette on upper left side) and stained for membrane localization of receptors 5-HT1b (green, a, e), 5-HT2a (green, c, g) and merged with actin, (red, b, d, f, h). Dotted line separates the near and far regions of the growth cone. i Increase magnification of representative image h to show the presence of 5-HT2a receptor membrane on most filopodia (*) of the growth cone (yellow *, insets). Scale bars are 5 μm. (j-k)There was no significant near/far bias in the amount of f-actin (/area) (j) and the number of filopodia (k) in growth cones exposed to vehicle and 5HT-Lo. Kruskal–Wallis, Dunn’s multiple comparison post hoc test. (l) There was no significant bias in the total percentage of filopodia containing 5HT2a and 5HT1b puncta (60%). One-way ANOVA followed by Tukey’s multiple comparison post hoc test. (m) There was no significant near/far bias in the amount of 5-HT2a and 5-HT1b puncta per filopodia. (n) Analysis of 5-HT receptor membrane localization showed translocation of 5-HT2a (n = 18, p = <0.0001) to the near side of growth cones exposed to 5HT-Lo while no significant translocation of 5-HT1b (n = 15, p = 0.346) was observed. (Mann–Whitney U-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Membrane, Staining, Comparison, Translocation Assay, MANN-WHITNEY

Journal: Frontiers in Psychiatry

Article Title: Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer’s disease

doi: 10.3389/fpsyt.2022.1020831

Figure Lengend Snippet: Effects of histone deacetylase (HDAC) inhibitors on histone acetylation at the 5htr2a and Drd2 gene promoters in the striatum in young and aged mice. (A) No significant differences of H3K27ac binding at the 5htr2a promoter between young and aged group was found, but a significant increase in H3K27ac binding at the 5ht2a promoter in the striatum was found in aged and young mice co-treated with RIS and VPA or MS-275 compared to those treated with RIS alone. (B) H3K27ac binding at the Drd2 promoter was significantly decreased in aged mice as compared to young mice in Veh treated group. However, H3K27ac binding at the Drd2 promoter in the striatum was observed to be significantly increased in aged mice co-treated with RIS and HDAC inhibitors VPA or MS-275 when compared to those treated with Veh alone. Data are presented as mean ± SEM, ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blots were blocked for 1-hour with 5% non-fat dry milk (Biorad, 1706404) and incubated using 1:1,000 5HT2A (Boster Biological, PA1373), 1:500 D2R (Millipore, AB5084P), or 1:1000 B-actin (Santa Cruz, sc-47778) overnight at 4°C.

Techniques: Histone Deacetylase Assay, Binding Assay

Journal: Atherosclerosis

Article Title: Specific Serotonin Receptor Antagonist Reduces Murine Calcific Atherosclerosis

doi: 10.1016/j.atherosclerosis.2026.120649

Figure Lengend Snippet: Representative images and quantitative bar graphs of (A) HTR-2A and (B) HTR-2B. Positive staining areas were normalized to total aortic area (Control, n = 7/group; Ketanserin, n = 7/group; LY272015 , n = 7/group). Magnification: 20X. Data were analyzed by Brown-Forsythe and Welch ANOVA test and Dunnett’s T3 multiple comparison tests, with individual variances computed for each comparison.

Article Snippet: Atherosclerosis was assessed by oil red O staining, and CD 68 (BioRad, CA), HTR-2A (Biorbyt, NC) and HTR-2B (Biorbyt, NC) were assessed by immunohistochemistry.

Techniques: Expressing, Staining, Control, Comparison