Journal: Advanced Science

Article Title: The IRE1‐XBP1s Axis Drives Inflammatory Osteolysis by Regulating a 5‐HT Dependent Endogenous Anti‐Autophagy Mechanism

doi: 10.1002/advs.76755

Figure Lengend Snippet: XBP1s activates the transcription of the 5‐HT transporter Slc6a4. (A) The chromosomal location, gene sequence, and the predicted XBP1s binding site within the promoter region of murine Slc6a4. (B) The schematic diagram of the chromosomal location, gene structure, and the predicted XBP1s binding site within the promoter region of human Slc6a4. (C) The binding motif of human XBP1s obtained from the JASPAR database. (D) The wild‐type and mutant sequence of the predicted human XBP1s binding site in the luciferase reporter constructs. (E) Dual luciferase activity of blank and overexpression human XBP1s in the wild‐type and mutant Slc6a4 promoter groups ( n = 3 per group). (F) ChIP‐qPCR results showing the binding of XBP1s to the promoter of Slc6a4. Homologous IgG antibody was used as a negative control in the ChIP assay, and the input lysis was used as the positive control in the PCR analysis. (G) GSEA of the 5‐HT uptake pathway between the control and 4μ8C‐treanted groups. (H) GSEA of the 5‐HT transport pathway between the control and 4μ8C‐treanted groups. (I) The level of 5‐HT in the supernatant of culture medium in the LPS and 4μ8C‐treanted groups ( n = 8 per group). (J) Intracellular 5‐HT levels in the LPS and 4μ8C‐treanted groups ( n = 6 per group). (K,L) Representative TRAP staining images (K) and quantification (L) of osteoclasts induced from BMDMs with Slc6a4 overexpression. Scale bars, 400 µ m . (M) The escitalopram (5‐HT transporter inhibitor) and asenapine (5‐HT receptor inhibitor) treatment on LPS‐mediated osteoclast differentiation in a similar concentration gradient manner. (N) Representative 3D reconstruction (micro‐CT) image of calvarium in each group. (O‐P) Quantification of bone mass parameters PAR (resorption pit area ratio) and RPC (Resorption pit count) ( n = 6 per group). (Q) Representative Trap staining images of calvarial slices from each group. Black arrows indicate osteoclasts. (R,S) Quantification of osteoclast‐related histomorphometric parameters N.OC/B.Pm and OC.S/B.S ( n = 6). Data were presents as mean ± SEM and the statistical significance was determined by two‐sided Student's test. Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: 5‐HT , MedChemExpress , Cat#HY‐B1473A.

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Construct, Activity Assay, Over Expression, ChIP-qPCR, Negative Control, Lysis, Positive Control, Control, Staining, Concentration Assay, Micro-CT

Journal: Advanced Science

Article Title: The IRE1‐XBP1s Axis Drives Inflammatory Osteolysis by Regulating a 5‐HT Dependent Endogenous Anti‐Autophagy Mechanism

doi: 10.1002/advs.76755

Figure Lengend Snippet: 5‐HT promotes LPS‐mediated osteoclast differentiation through endogenic anti‐autophagy mechanism. (A) The principal component analysis of the RNA‐sequencing data in the control and 5‐HT‐treated groups. (B) Volcano plot shows that 5‐HT upregulates 249 DEGs, including IL‐1β and TNFα. (C) KEGG pathway analysis of the 249 5‐HT‐upregulated genes. (D) Representative TRAP staining images of mature osteoclasts with different TNFα stimulation. Scale bars: 400 µm. (E) mRNA expression of osteoclast‐associated genes in BMDMs with or without TNFα stimulation. (F) Representative TRAP staining images of mature osteoclasts with different IL‐1β stimulation. Scale bars: 400 µm. (G) The design of non‐targeted metabolomics analysis with or without the treatment of 5‐HT. (H) Volcano plot shows 152 reduced metabolites with the intervention of 5‐HT. (I) The heatmap displays primarily downregulated and upregulated metabolites under 5‐HT treatment. (J) The total ion chromatogram (TIC) displays the retention time (RT) of the 3MA standard peak under multiple reaction monitoring (MRM), used to evaluate the mass spectrometry (MS) conditions. (K) The mass‐to‐charge ratio (m/z) range and RT for 3MA were set as the MS conditions, under which 3MA could be clearly detected with observable peak formation in the extracted ion chromatogram (EIC). (L) In the EIC, the peak area of 3MA showed a linear correlation with its standard concentration (R = 0.9922), and the signal‐to‐noise ratio (S/N) was ≥107.98, meeting the quantitative analysis requirements for experimental samples. (M) Quantitative analysis shows the mass spectrometry detection results of intracellular 3MA with or without 5‐HT stimulation (N = 6). (N) Mass spectrometry detection and quantitative analysis of intracellular 3MA levels with or without escitalopram stimulation (N = 5). Data were presented as mean ± SEM, and the statistical significance was determined by two‐sided Student's test. Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: 5‐HT , MedChemExpress , Cat#HY‐B1473A.

Techniques: RNA Sequencing, Control, Staining, Expressing, Targeted Proteomics, Mass Spectrometry, Concentration Assay

Journal: Advanced Science

Article Title: The IRE1‐XBP1s Axis Drives Inflammatory Osteolysis by Regulating a 5‐HT Dependent Endogenous Anti‐Autophagy Mechanism

doi: 10.1002/advs.76755

Figure Lengend Snippet: 3MA suppresses 5‐HT‐mediated enhancement of cellular autophagy. (A) Representative TRAP staining images of mature osteoclasts with or without 5‐HT and 3MA stimulation. Scale bars: 400 µm. (B) qPCR result shows the expression of TNFα with the supplementation of 3MA. (C) ELISA analysis shows the TNFα level in the culture medium supernatant. (D) Protein levels of autophagy‐special genes in the BMDMs with or without the supplementation of 3MA. (E) Representative 3D reconstruction (micro‐CT) images of calvarium in each group. (F,G) Quantification of bone mass parameters PAR (F) and RPC (G) (n = 6 per group). (H) Representative Trap staining images of calvarial slices from each group. Black arrows indicate osteoclasts. (I,J) Quantification of osteoclast‐related histomorphometric parameters OC.S/B.S and N.OC/B.Pm ( n = 6 per group). (K) Immunofluorescence of CTSK‐labeled calvarial slices from each group. (L‐N) Representative images of TRAP staining (L) and quantification of PAR (M) and RPS (N) in healthy donors and myelitis patient groups ( n = 6 per group). Scale bars: 200 µm. (O–Q) Representative TRAP staining images in mature human osteoclasts induced from peripheral blood mononuclear cells (PBMC) of healthy donors, myelitis (O), RA (P), and OA (Q) patients. Scale bars: 200 µm. Data were presented as mean ± SEM, and the statistical significance was determined by two‐sided Student's test. Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: 5‐HT , MedChemExpress , Cat#HY‐B1473A.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Micro-CT, Immunofluorescence, Labeling

Journal: Advanced Science

Article Title: The IRE1‐XBP1s Axis Drives Inflammatory Osteolysis by Regulating a 5‐HT Dependent Endogenous Anti‐Autophagy Mechanism

doi: 10.1002/advs.76755

Figure Lengend Snippet: Schematic diagram of the molecular mechanism of inflammatory osteolysis. A graphical abstract for the mechanisms by which IRE1‐XBP1s‐Slc6a4 axis regulates inflammatory osteoclast differentiation through a 5‐HT dependent endogenous anti‐autophagy. The bacterial or sterile inflammation promotes macrophages releasing proinflammatory factors, which can trigger the UPR in osteoclast precursor cells and active the peripheral inflammatory response. IRE1 converts XBP1u to XBP1s by unconventional splicing, which can translocalize to the nucleus and bind to the Slc6a4 promoter to regulate its expression. The 5‐HT transporter, encoded by Slc6a4, colonized the cell membrane and reuptakes the peripheral‐derived 5‐HT driven by inflammation into the cell. By inhibiting 3MA anabolism, 5‐HT can weaken the inherent anti‐autophagy ability of cells and then promote osteoclast differentiation.

Article Snippet: 5‐HT , MedChemExpress , Cat#HY‐B1473A.

Techniques: Sterility, Expressing, Membrane, Derivative Assay

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A – D ) RT-qPCR analysis of CDKN2A , CDKN1A , TP53 , IL1B , and IL6 mRNA expression in G608G MSC with or without metabolite treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. P values from left to right: ( A ) P = 0.04, P = 0.002, P = 0.46, P = 0.03, P < 0.001; ( B ) P = 0.45, P < 0.001, P = 0.18, P < 0.001, P = 0.01; ( C ) P = 0.05, P = 0.006, P < 0.001, P = 0.34, P < 0.001; ( D ) P = 0.83, P < 0.001, P = 0.009, P < 0.001, P < 0.001. n = 3 biological repeats. ( E , F ) Immunofluorescence analysis ( E ) and quantification ( F ) of 5-HT of 4-week-old C57 and 12-month-old C57 MII oocytes. Scale bar, 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-tailed unpaired t test. ( F ) P = 0.01. n = 10–11 biological repeats. ( G ) Bright-field imaging depicting the development status of GV from 4-week-old C57 mice and 12-month-old C57 mice treated with 5-HT or not. The arrowheads point to MII. Scale bar, 100 μm. ( H ) The proportion of GV, MI, and MII is analyzed in the form of overlapping graphs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. P = 0.005, and P < 0.001. The analysis specifically focused on MII. n = 3 biological repeats. ( I – K ) Immunofluorescence analysis ( I ) of MitoTracker and DCFH-DA-based ROS levels in MII oocytes developed from GV of oocyte from 4-week-old and 12-month-old mice treated with or without 5-HT in vitro. Quantitative analysis of the fluorescence intensity of mitochondria ( J ) and the ROS level ( K ). Scale bar, 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( J ) P = 0.69, and P = 0.02; ( K ) P = 0.04, and P = 0.01. n = 8–9 biological repeats. ( L , M ) Immunofluorescence analysis ( L ) of tubulin in oocytes stage (developed from GV in vitro) of 4-week-old and 12-month-old C57 mice treated with or without 5-HT in vitro. Quantitative analysis ( M ) of normal/abnormal spindle ratio of MII. Scale bar, 20 μm. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Two Tailed Test, Imaging, In Vitro, Fluorescence

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A , B ) SA-β-Gal staining ( A ) and quantification ( B ) of WT MSC and G608G MSC with or without 5-HT treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( B ) P = 0.001, and P = 0.01. n = 3 biological repeats. Scale bar, 100 μm. ( C ) CCK-8 evaluation of proliferation of WT MSC and G608G MSC with or without 5-HT treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. WT vs G608G: P < 0.001; G608G vs G608G + 5-HT: P < 0.001. n = 3 biological repeats. ( D ) RT-qPCR analysis of CDKN2A , CDKN1A , TP53 , IL6 , and IL1B mRNA expression in WT MSC and G608G MSC with or without 5-HT treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.002, P = 0.03; P < 0.001, P = 0.003; P = 0.16, P = 0.96; P = 0.002, P = 0.05; P = 0.002, P = 0.009. n = 3 biological repeats. ( E , F ) SA-β-Gal staining ( E ) and quantification ( F ) of young generations of WT MSC(WT Y), old generations of WT MSC(WT O), and old generations of WT MSC treated with 5-HT(WT O + 5-HT, 5 μM). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( F ) P < 0.001, and P = 0.001. n = 3 biological repeats. Scale bar, 100 μm. ( G ) CCK-8 evaluation of the proliferation of WT Y, WT O, and WT O + 5-HT. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. WT Y vs WT O: P < 0.001; WT O vs WT O + 5-HT: P < 0.001. n = 3 biological repeats. ( H ) RT-qPCR analysis of CDKN2A , CDKN1A , TP53 , IL6 , and IL1B mRNA expression of WT Y, WT O, and WT O + 5-HT. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.01, P = 0.02; P < 0.001, P < 0.001; P < 0.001, P < 0.001; P < 0.001, P = 0.04; P < 0.001, P < 0.001. n = 3 biological repeats. ( I , J ) Cell cycle distribution patterns of WT MSCs and G608G MSCs of identical passage number ( I ) and young and aged WT MSCs ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. ( I ) P < 0.001, and P < 0.001; ( J ) P = 0.002, and P < 0.001. n = 3 biological repeats. ( K ) Meier survival plot of G608G mice treated with 0.9% NaCl ( n = 22) or 5-HT ( n = 23). The Log-rank (Mantel–Cox) test was used for statistical analysis. P = 0.0007; * P < 0.05, ** P < 0.01, *** P < 0.001. ( L – O ) Masson staining ( L ) and quantification of fibrosis area ( M – O ) of lung, muscle, and aorta in WT and G608G mice treated with 5-HT or not. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( M ) P < 0.001, and P = 0.003; ( N ) P = 0.04, and P = 0.02; ( O ) P = 0.002, and P = 0.006. n = 3 biological repeats. Scale bar, 200 and 100 μm. ( P – S ) Masson staining ( P ) and quantification of fibrosis area ( Q – S ) of lung, muscle, and aorta in 4-week-old and 20-month-old mice treated with 5-HT or not (from 12-month-old to 20-month-old). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( Q ) P = 0.009, and P = 0.03; ( R ) P = 0.001, and P = 0.003; ( S ) P < 0.001, and P = 0.01. n = 3–4 biological repeats. Scale bar, 200, 50 and 100 μm. Data are presented as mean ± SD. See also Fig. . .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Staining, CCK-8 Assay, Quantitative RT-PCR, Expressing

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A – C ) Immunofluorescence analysis ( A ) and quantification of γ-H2AX ( B ) and NCL ( C ) in WT MSC and G608G MSC with or without 5-HT treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( B ) P < 0.001, and P < 0.001. n = 3 biological repeats. Scale bar, 10 μm. ( D , E ) Open-field trajectory diagram ( D ) of 5-month-old WT and G608G mice treated with 5-HT or not (from 1-month-old to 5-month-old). Quantitative analysis of the total distance covered ( E ). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( E ) P = 0.05, and P = 0.03. n = 3 biological repeats. ( F , G ) Quantitative analysis of the average pulling strength of male ( F ) and female ( G ) 5-month-old WT and G608G mice treated with 5-HT or not, * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( F ) P = 0.003, and P = 0.09; ( G ) P < 0.001, and P < 0.001. n = 3 biological repeats. ( H – K ) Histological analysis (HE) ( H ) of the liver, spleen, and aorta of 5-month-old WT and G608G mice treated with 5-HT or not; Quantitative analysis of the number of central veins per unit area (mm 2 ) in the liver ( I ), the average VSMC number per unit area (mm 2 ) in the aorta ( J ), and the percentage of germinal center area in the spleen ( K ). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( I ) P < 0.001, and P = 0.006; ( J ) P = 0.008, and P = 0.05; ( K ) P < 0.001, and P = 0.003. n = 5–6 biological repeats. Scale bar, 100, 200 and 50 μm. ( L ) HE section of the ovary of 4-month-old WT and G608G mice treated with 5-HT or not. Scale bar, 200 and 50 μm. ( M ) Quantitative analysis of the number of follicles in each layer of ovaries of G608G mice treated with 5-HT or not. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.008, and P = 0.04; P = 0.03, and P = 0.44; P = 0.007, and P = 0.8; P < 0.001, and P = 0.04. n = 3 biological repeats. ( N – Q ) Histological analysis (HE) ( N ) of the liver, spleen, and aorta of 4-week-old and 20-month-old C57 mice treated with 5-HT or not (from 12-month-old to 20-month-old); Quantitative analysis of the number of central veins per unit area (mm 2 ) in the liver ( O ), the average VSMC number per unit area (mm 2 ) in the aorta ( P ), and the percentage of germinal center area in the spleen ( Q ). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( O ) P = 0.02, and P = 0.003; ( P ) P < 0.001, and P = 0.94; ( Q ) P = 0.009, and P = 0.009. n = 3–4 biological repeats. Scale bar, 200 and 50 μm. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Immunofluorescence

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A , B ) RT-qPCR analysis of Il1b , Il12 and Il6 mRNA expression of liver and kidney in naturally aged and G608G mice treated with 5-HT or not. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: ( A ) Kidney: P = 0.02, and P = 0.64; P = 0.77, and P = 0.95; P = 0.01, and P = 0.72. Liver: P = 0.12, and P = 0.43; P = 0.04, and P = 0.12; P = 0.03, and P = 0.04. ( B ) Kidney: P = 0.05, and P = 0.28; P = 0.03, and P = 0.06; P = 0.04, and P = 0.03. Liver: P = 0.65, and P = 0.05; P = 0.21, and P = 0.42; P = 0.03, and P = 0.37. n = 3–4 biological repeats. ( C , D ) RT-qPCR analysis of Cdkn2a and Cdkn1a mRNA expression of liver and kidney in naturally aged and G608G mice treated with 5-HT or not. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( C ) Kidney: P = 0.04, and P = 0.04; P = 0.002, and P = 0.04. Liver: P = 0.03, and P = 0.04; P = 0.43, and P = 0.41. ( D ) Kidney: P = 0.03, and P = 0.01; P = 0.006, and P = 0.05. Liver: P = 0.005, and P = 0.05; P = 0.005, and P = 0.002. n = 3–4 biological repeats. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Quantitative RT-PCR, Expressing

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of the differentially expressed genes ( P value ≤ 0.05, |log2Foldchange | ≥1) between WT MSC and G608G MSC and between G608G MSC and G608G MSC treated with 5-HT, Red: G608G vs WT UP, Green: G608G + 5-HT vs G608G DOWN, Blue: G608G + 5-HT vs G608G UP, Purple: G608G vs WT DOWN. ( B ) Genome-wide KEGG pathways analysis of upregulated and downregulated genes between G608G MSC and WT MSC and between G608G MSC and G608G + 5-HT. KEGG pathway enrichment was performed by over-representation analysis using a hypergeometric test (Fisher’s exact test), followed by Benjamini–Hochberg FDR correction (adjusted P values/ q values). ( C ) Western blots of PGC1α, TOMM20, and TIMM23 in WT MSC, G608G MSC, and G608G MSC treated with 5-HT. Lower: Quantification of the protein levels relative to β-actin. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. PGC1α: P < 0.001, and P = 0.04; TOMM20: P = 0.001, and P = 0.03; TIMM23: P = 0.006, and P = 0.04. n = 3 biological repeats. ( D ) The ATP levels of WT MSC, G608 MSC, and G608G were treated with 5-HT. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P = 0.008, and P = 0.01. n = 3 biological repeats. ( E – G ) A representative seahorse plot ( E ) of WT MSC, G608G MSC, and 5-HT-treated G608G MSC, with measurements of OCR (normalized to protein), and following treatment of cells with oligomycin (Olig), fluorcarbonylcyanide phenylhydrazone (FCCP), and rotenone (Rot) as indicated. Quantitative analysis of the effects of 5-HT on basal respiration ( F ) and maximal respiration ( G ) (compiled results from n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( F ) P < 0.001, and P = 0.003; ( G ) P < 0.001, and P = 0.03. n = 4 biological repeats. ( H , I ) Immunofluorescence experiment of MitoTracker ( H ) and fluorescence intensity quantification in analysis ( I ) of WT MSC, G608G MSC, and 5-HT-treated G608G MSC. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. ( I ) P < 0.001 and P = 0.03. n = 4 biological repeats. Scale bar, 10 μm. ( J ) Schematics showing that 5-HT promote mitochondrial regeneration and regulate mitochondrial function. Data are presented as mean ± SD. See also Fig. . .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Genome Wide, Western Blot, Immunofluorescence, Fluorescence

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A – C ) Heatmaps of the genes related to inflammation ( A ), mitochondria ( B ), and proliferation ( C ) of WT MSC and G608G MSC treated with 5-HT or not. ( D ) RT-qPCR analysis of 5-HT receptor subtypes in MSC. n = 3 biological repeats. ( E ) Heatmap of expression levels of all 5-HT receptors in MSC from RNA sequencing results mentioned above. ( F ) CCK-8 evaluation of proliferation of G608G MSC treated with 5-HT or 5-HT + SB (SB: SB-224289 hydrochloride). * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. G608G vs G608G + 5-HT: P < 0.001. G608G + 5-HT vs G608G + 5-HT + SB: P < 0.001. n = 3 biological repeats. ( G ) RT-qPCR analysis of CDKN2A , CDKN1A , TP53 , IL6 and IL1B mRNA expression in G608G MSC treated with 5-HT or 5-HT + SB. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.69, and P = 0.93; P = 0.02, and P < 0.001; P = 0.01, and P = 0.03; P = 0.02, and P = 0.001; P = 0.42, and P < 0.001. n = 3 biological repeats. ( H ) Western blots of PGC1α, TOMM20, and TIMM23 protein levels in G608G MSC treated with 5-HT or 5-HT + SB. Right: Quantification of the protein levels relative to β-actin. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. PGC1α: P < 0.001, P = 0.01, and P = 0.03; TOMM20: P = 0.01, P = 0.03, and P < 0.001; TIMM23: P = 0.003, P = 0.03, and P = 0.03. n = 3 biological repeats. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing, CCK-8 Assay, Western Blot

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A , B ) GO analysis of upregulated and downregulated genes between G608G MSC and WT MSC ( A ) and between G608G MSC with or without 5-HT treatment ( B ). GO enrichment was performed by over-representation analysis using a hypergeometric test (Fisher’s exact test), followed by Benjamini–Hochberg FDR correction (adjusted P values/q values). ( C ) Workflow for the discovery of serotonylated proteins by LC-MS/MS and IP. ( D ) Heatmap of the top 20 enriched proteins. ( E ) MSCs were cultured 72 h, with or without 5 μmol/L 5-PT. 5-PT was immunoprecipitated by beads, and HSP90β were detected. IP immunoprecipitation. Upper: G608G MSCs, Lower: WT MSCs. ( F ) RT-qPCR analysis of CDKN2A , CDKN1A , and TP53 mRNA expression in MSCs treated with 5-HT, LDN or LDN + 5-HT, LDN: LDN-27219, TGM2 inhibitor. Upper: G608G MSCs, Lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. G608G vs G608G + 5-HT: CDKN2A : P = 0.03; CDKN1A : P = 0.02; TP53 : P < 0.001. WT vs WT + 5-HT: CDKN2A : P < 0.001; CDKN1A : P = 0.002; TP53 : P < 0.001. WT + LDN vs WT + LDN + 5-HT: CDKN2A : P < 0.001. n = 3 biological repeats. ( G ) SA-β-Gal staining (upper) and quantification (lower) of MSCs treated with LDN or LDN + 5-HT. Left: G608G MSCs, Right: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. G608G vs G608G + 5-HT: P = 0.03; G608G vs G608G + LDN: P = 0.003. WT vs WT + 5-HT: P = 0.006. n = 3 biological repeats. Scale bar, 100 μm. Data are presented as mean ± SD. See also Fig. . .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Immunoprecipitation, Quantitative RT-PCR, Expressing, Staining

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) Immunofluorescence analysis (left) and quantification of Ki67 (right) in MSCs treated with 5-HT, LDN or LDN + 5-HT. upper: G608G MSCs, lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. G608G vs G608 + 5-HT: P = 0.004; G608G vs G608G + LDN: P < 0.001. WT vs WT + 5-HT: P = 0.002; WT vs WT + LDN: P < 0.001. n = 3 biological repeats. Scale bar, 5 μm. ( B ) RT-qPCR analysis of HSP90AB1 mRNA expression in MSCs transfected with control shRNA or shRNA against HSP90AB1. Left: G608G MSCs, Right: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-tailed unpaired t test. P < 0.001 (left), and P < 0.001 (right). n = 3 biological repeats. ( C ) Immunofluorescence analysis (left) and quantification of Ki67 (right) in MSCs transfected with control shRNA or shRNA against HSP90AB1. upper: G608G MSCs, lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-tailed unpaired t test. P = 0.004 (upper), and P = 0.004 (lower). n = 3 biological repeats. Scale bar, 5 and 10 μm. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Immunofluorescence, Quantitative RT-PCR, Expressing, Transfection, Control, shRNA, Two Tailed Test

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) RT-qPCR analysis CDKN2A , CDKN1A , and TP53 mRNA expression in MSCs transfected with control shRNA or shRNA against HSP90AB1. Left: G608G MSCs, Right: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-way ANOVA test. P values from left to right: G608G MSCs: P = 0.002, P < 0.001, P < 0.001; WT MSCs: P = 0.02, P < 0.001, P = 0.004. n = 3 biological repeats. ( B ) Western blots of HSP90β, and CHOP protein levels in MSCs transfected with control shRNA or shRNA against HSP90AB1. Left: G608G MSCs, Right: WT MSCs. Lower: Quantification of the protein levels relative to β-actin. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-tailed unpaired t test. HSP90β: P = 0.002 (left), and P = 0.02 (right); CHOP: P = 0.004 (left), and P < 0.001 (right). n = 3 biological repeats. ( C ) SA-β-Gal staining (left) and quantification (right) of MSCs transfected with control shRNA or shRNA against HSP90AB1. Upper: G608G MSCs, Lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the two-tailed unpaired t test. P < 0.001 (upper), and P < 0.001 (lower). n = 3 biological repeats. Scale bar, 100 μm. ( D ) LC-MS/MS analysis at Gln207 of TGM2-transamidated serotonin to full-length HSP90β, serotonylation peptide (top), unserotonylation peptide (below). ρ = Ser-NH3 is the difference in mass-charge ratio between b5 in the top and b5 in the bottom and between b7 in the top and b7 in the bottom, where the difference in mass-charge ratio is 159. ( E ) The domain of HSP90β and the representation of the serotonylation site. ( F ) shHSP90AB1 G608G MSCs were transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala(Q207A, Q493A) mutated HSP90β. Cells were treated with 5-HT or not. DDIT3 , ATF4 , and PPP1R15A mRNA expression was detected. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: DDIT3: P = 0.01, and P = 0.01; P < 0.001, and P = 0.26; P = 0.003, and P < 0.001. ATF4: P = 0.002, and P < 0.001; P = 0.99, and P = 0.27; P = 0.02, and P < 0.001. PPP1R15A: P = 0.008, and P < 0.001; P = 0.08, and P = 0.97; P = 0.006, and P = 0.004. n = 3 biological repeats. Data are presented as mean ± SD. See also Figs. and . .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, shRNA, Western Blot, Two Tailed Test, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) RT-qPCR analysis of HSP90AB1 mRNA expression in MSCs and HSP90AB1-overexpression MSCs with or without 5-HT treatment. Left: G608G MSCs, Right: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P < 0.001, and P < 0.001; P < 0.001, and P < 0.001. n = 3 biological repeats. ( B ) RT-qPCR analysis of CDKN2A , TP53 , and IL1B or CDKN1A mRNA expression in MSCs and HSP90AB1-overexpression MSCs with or without 5-HT treatment. Upper: G608G MSCs, Lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: G608G MSCs: P < 0.001, and P < 0.001; P = 0.005, and P = 0.04; P = 0.002, and P < 0.001. WT MSCs: P < 0.001, and P = 0.003; P < 0.001, P = 0.02; P < 0.001, and P = 0.02. n = 3 biological repeats. ( C ) Western blots of HSP90β, CHOP protein levels in MSCs and HSP90AB1-overexpression MSCs with or without 5-HT treatment. Left: G608G MSCs, Right: WT MSCs. Lower: Quantification of the protein levels relative to β-actin. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. HSP90β: P < 0.001, and P < 0.001 (left); P = 0.01, and P = 0.01 (right).CHOP: P = 0.03, and P = 0.004 (left); P < 0.001, P = 0.03 (right). n = 3 biological repeats. ( D ) SA-β-Gal staining (left) and quantification (right) of MSCs and HSP90AB1-overexpression MSCs with or without 5-HT treatment. Upper: G608G MSCs, Lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P < 0.001, and P = 0.002 (upper); P < 0.001, and P = 0.002 (lower). n = 3 biological repeats. Scale bar, 100 μm. ( E ) Immunofluorescence analysis (left) and quantification of Ki67 (right) in MSCs and HSP90AB1-overexpression MSCs with or without 5-HT treatment. upper: G608G MSCs, lower: WT MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P = 0.002, and P < 0.001 (upper); P < 0.001, and P < 0.001 (lower). n = 3 biological repeats. Scale bar, 5 μm. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Staining, Immunofluorescence

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) LC-MS/MS analysis at Gln493 of TGM2-transamidated serotonin to full-length HSP90β, serotonylation peptide (top), unserotonylation peptide (below). ρ = Ser-NH3 is the difference in mass-charge ratio between two parent ion, where the difference in mass-charge ratio is 159. ( B ) The ATPase activity of HSP90β. Fluorescence was measured at λex 485 nm, λex 530 nm using a Bio-Tek fluorescent microplate reader. ( C ) shHSP90AB1 G608G MSCs were transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala(Q207A, Q493A) mutated HSP90β. Cells were treated with 5 μM 5-PT or not. 5-PT was immunoprecipitated by beads, and Flag-HSP90β were detected. IP immunoprecipitation. ( D ) shHSP90AB1 G608G MSCs were transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala(Q207A, Q493A) mutated HSP90β. Cells were treated with 5-HT or not. IL1B , CDKN1A and CDKN2A mRNA expression was detected. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: IL1B : P = 0.01, and P = 0.02; P = 0.005, and P = 0.08; P < 0.001, and P = 0.005. CDKN1A : P < 0.001, and P < 0.001; P < 0.001, and P = 0.13; P < 0.001, and P = 0.01. CDKN2A : P = 0.03, and P = 0.007; P = 0.85, and P = 0.04; P = 0.03, and P = 0.004. n = 3 biological repeats. ( E ) SA-β-Gal staining (upper) and quantification (lower) of shHSP90AB1 G608G MSCs transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala (Q207A, Q493A) mutated HSP90β, cells were treated with 5-HT or not. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P < 0.001, and P = 0.003; P = 0.006, and P = 0.93; P < 0.001, and P = 0.002. n = 3–4 biological repeats. Scale bar, 100 μm. ( F ) Schematics showing that 5-HT regulates cellular aging through both classical and non-classical functions, by Figdraw. Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Liquid Chromatography with Mass Spectroscopy, Activity Assay, Fluorescence, Transfection, Expressing, Immunoprecipitation, Staining

Journal: The EMBO Journal

Article Title: The oocyte-enriched metabolite serotonin alleviates cellular senescence and aging phenotypes in the mouse

doi: 10.1038/s44318-026-00832-x

Figure Lengend Snippet: ( A ) shHSP90AB1 G608G MSCs were transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala(Q207A, Q493A) mutated HSP90β. Cells were treated with 5-HT or not. Immunofluorescence analysis and quantification of Fura-2 AM. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.04, and P = 0.04; P = 0.04, and P = 0.83; P = 0.002, and P = 0.005. n ≥ 3 biological repeats. Scale bar, 20 μm. ( B ) shHSP90AB1 WT MSCs were transfected with mRFP-fused lentiviruses expressing either WT-HSP90β or Gln to Ala(Q207A, Q493A) mutated HSP90β. Cells were treated with 5-HT or not. Immunofluorescence analysis and quantification of Fura-2 AM. * P < 0.05, ** P < 0.01, *** P < 0.001 according to the one-way ANOVA test. P values from left to right: P = 0.01, and P = 0.03; P = 0.002, and P = 0.81; P < 0.001, and P < 0.001. n ≥ 3 biological repeats. Scale bar, 20 μm. ( C ) Schematics showing that 5-HT induced HSP90β Q207ser serotonylation and reduced ER stress in MSC cells (by Figdraw). Data are presented as mean ± SD. .

Article Snippet: In brief, serotonylated proteins were labeled by using copper click chemistry, Alkyne-functionalized 5-HT derivative 5-PT was conjugated to a biotin–azide molecule and prepared in MedChemExpress (MCE®), cells were treated with 10 μM 5-PT for 72 h. Cell pellets were sonicated in 100 μL CLICK reaction buffer (Thermo, C10643 ), then centrifuged at 15,000 rpm *15 min at 4 °C, supernatant was then transferred to a new 1.5 ml EP tube.

Techniques: Transfection, Expressing, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Polygonatum sibiricum combined with Poria cocos prevents spleen deficiency constipation via the gut microbiota–mediated brain–gut axis

doi: 10.3389/fphar.2026.1793441

Figure Lengend Snippet: Serum levels of 5-HT, CCK, VIP, and MTL in mice from each group. (A) 5-HT content; (B) CCK content; (C) MTL content; (D) VIP content. Note: NC: normal control group; NM: model group; HF: Polygonatum sibiricum and Poria cocos group.

Article Snippet: Mouse Serotonin (5-HT) ELISA Research Kit (Cat# JM-02726M2), Mouse Cholecystokinin (CCK) ELISA Research Kit (Cat# JM-02311M2), Mouse Motilin (MTL) ELISA Research Kit (Cat# JM-02775M2), Mouse Vasoactive Intestinal Peptide (VIP) ELISA Research Kit (Cat# JM-02729M2), the aforementioned kits were procured from Jiangsu Jingmei Biotechnology Co., Ltd. Superoxide Dismutase (SOD) Activity Assay Kit (BC0175), Malondialdehyde (MDA) Content Assay kit (BC0025), the aforementioned kits were procured from Beijing Solarbio Science & Technology Co., Ltd.

Techniques: Control

Journal: Research

Article Title: Platelets Orchestrate a Neuroimmune Axis Driving Cutaneous Inflammation and Itch

doi: 10.34133/research.1294

Figure Lengend Snippet: Platelet-derived serotonin contributes to itch transmission and macrophage accumulation in AD. (A) Cutaneous 5-HT concentrations measured by ELISA in photostimulated Ai32 and PF4 Cre ; Ai32 mice. n = 4 photostimulated Ai32 mice. n = 6 photostimulated PF4 Cre ; Ai32 mice. t 8 = 3.900, P = 0.0045 (2-tailed unpaired Student’s t test). (B) Skin 5-HT concentrations measured by ELISA in the vehicle- and MC903-treated mice. n = 4 mice per group. t 6 = 17.93, P < 0.0001 (2-tailed unpaired Student’s t test). (C) Skin 5-HT concentrations measured by ELISA in the platelet-depleted PF4 Cre ; iDTR mice and control littermates after MC903 treatment. n = 4 mice per group. t 6 = 8.558, P = 0.0001 (2-tailed unpaired Student’s t test). (D) Schematic drawing of peripheral 5-HT synthesis and release pathway. (E) Representative images of ears from WT and TPH1 −/− after MC903 treatment. Scale bar, 1 mm. (F and G) Ear thickness (F) and inflammation score (G) measured at the indicated time points. For (F), F 6,56 = 4.830, P = 0.0005. Day 7: P < 0.0001, day 9: P < 0.0001, day 11: P = 0.0022. For (G), F 6,56 = 5.066, P = 0.0003. Day 7: P < 0.0001, day 9: P < 0.0001, day 11: P = 0.0120 (2-way ANOVA followed by Šídák’s multiple comparisons test). (H) Quantification of spontaneous scratching bouts in WT and TPH1 −/− after MC903 treatment. n = 5 mice per group. F 6,56 = 3.936, P = 0.0026. Day 7: P < 0.0001, day 9: P = 0.0017, day 11: P < 0.0001 (2-way ANOVA followed by Šídák’s multiple comparisons test). (I) Representative H&E histopathology images of ears. n = 3 to 5 sections from 3 mice. Scale bar, 100 μm. (J and K) Quantification of dermal thickness (J) and dermal immune cell infiltration (K). n = 5 mice per group. For (J), t 8 = 2.935, P = 0.0188. For (K), t 8 = 5.305, P = 0.0007 (2-tailed unpaired Student’s t test). (L and M) Representative traces of spontaneous C-fiber firings recorded ex vivo, quantified in (M). n = 6 units from 4 mice. t 10 = 8.941, P < 0.0001 (2-tailed unpaired Student’s t test). (N and O) Representative images of F4/80 + macrophages in the skin of MC903-treated WT and TPH1 −/− mice, which were quantified in (O). Scale bar, 50 μm. n = 4 mice per group. For (O), t 6 = 6.849, P = 0.0005 (2-tailed unpaired Student’s t test). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Individual data points in (M) represent single animals, and bars show mean ± SD from 2 independent experiments. Result in (A) to (C), (F) to (H), (J), (K), and (O), individual data points represent single animals and are shown as mean ± SD from one representative of 2 independent experiments with consistent results.

Article Snippet: To assess the pruritogenic and pro-inflammatory effects of 5-HT in vivo, mice were subjected to acute or repeated intradermal 5-HT (HY-B1473, 100 μM, 20 μl, MCE, NJ, USA) administration in the ear pinna [ ].

Techniques: Derivative Assay, Transmission Assay, Enzyme-linked Immunosorbent Assay, Control, Histopathology, Ex Vivo