Journal: Cell

Article Title: Serotonin transporter inhibits antitumor immunity through regulating the intratumoral serotonin axis

doi: 10.1016/j.cell.2025.04.032

Figure Lengend Snippet: (A) Schematics showing the proposed autocrine serotonin signaling pathway in a CD8 T cell. Possible pharmacological interventions are indicated. (B and C) Serotonergic gene expression in Sert -WT CD8 T cells in response to antigen stimulation. Naive CD8 T cells were purified from Sert -WT B6 mice and stimulated with anti-CD3 for 3 days. (B) RT-qPCR analyses of 5-HTR family member gene expression at day 1 ( n = 4). Unstimulated naive CD8 T cells were included as a control. (C) RT-qPCR analyses of Sert , Tph1 , and Maoa gene expression over time ( n = 4). (D–H) Serotonergic gene expression in activated Sert -WT and Sert -KO CD8 T cells. (D) Experimental design. Naive CD8 T cells were purified from Sert -WT and Sert -KO B6 mice and stimulated in vitro with anti-CD3 for 1 day. (E–H) RT-qPCR analyses of Tph1 (E), Maoa (F), Htr2b (G), and Htr7 (H) expression ( n = 4). (I–M) Autocrine serotonin signaling in Sert -WT CD8 T cells. (I) Experimental design. Sert -WT CD8 T cells were stimulated with anti-CD3 for 3 days in serotonin-depleted medium in the presence or absence of SSRI (FLX or CIT) and/or 5-HTR antagonist (ASE or RS-127445) treatment. ASE, asenapine (a general antagonist of most 5-HTR subtypes); RS-127445, a 5-HTR2B selective antagonist. (J and K) RT-qPCR analyses of Il2 (J) and Ifng (K) expression in FLX-treated or non-treated (NT) CD8 T cells at day 2, with or without ASE treatment ( n = 3). (L) RT-qPCR analyses of Il2 expression in FLX-treated or non-treated (NT) CD8 T cells at day 2, with or without RS-127445 treatment ( n = 3). (M) ELISA analyses of serotonin levels over time in culture supernatants of FLX-treated, CIT-treated, or non-treated (NT) CD8 T cells ( n = 4). (N and O) Western blot analyses of key signaling molecules involved in the 5-HTR-MAPK (N) and TCR (O) signaling pathways. MAPK, mitogen-activated protein kinase. (P–R) Serotonin levels in Sert -WT and Sert -KO BMT mice bearing B16-OVA tumors (denoted as WT and KO, respectively). (P) Experimental design. (Q and R) HPLC analyses of serotonin levels in tumor (Q, n = 4) and serum (R, n = 5–6) at day 21. (S–U) Serotonin levels in Sert -WT mice bearing B16-OVA tumors, with or without SSRI (i.e., FLX) and/or anti-CD8 depletion antibody treatment. (S) Experimental design. (T and U) HPLC analyses of serotonin levels in tumor (T, n = 7–8) and serum (U, n = 4–5) at day 14. Representative of two (B, E–H, J–M, N, and O) and three (C, Q, R, T, and U) experiments. Data are presented as the mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t test (B, E–H, Q, and R), one-way ANOVA (C and M), or two-way ANOVA with Turkey’s multiple comparisons test (J–L, T, and U). See also .

Article Snippet: C57BL/6J (B6), BALB/cJ (BALB/c), B6.SJL- Ptprc a Pepc b /BoyJ (CD45.1), C57BL/6-Tg (TcraTcrb)1100Mjb/J ( OT1 -Tg), B6.129(Cg)- Slc6a4 tm1Kpl /J ( Sert -KO), B6.129- Htr7 tm1Sut /J ( 5Htr7 -KO), and NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NOD scid gamma or NSG) mice were purchased from the Jackson Laboratory (JAX; Bar Harbor).

Techniques: Gene Expression, Purification, Quantitative RT-PCR, Control, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Protein-Protein interactions

Journal: International journal of molecular sciences

Article Title: Restraint Stress Disrupted Intestinal Homeostasis via 5-HT/HTR7/Wnt/β-Catenin/NF-kB Signaling.

doi: 10.3390/ijms26094021

Figure Lengend Snippet: Figure 1. Restraint stress induced an increase in intestinal 5-HT levels. (a) Changes in 5-HT levels in serum and intestine of mice (n= 5). (b,c) Changes in Tph1 and Tph2 mRNA levels in jejunum and colon tissues of mice (n = 10). (d) EC+ cells in the jejunum and colon were detected by immunohisto- chemistry, and (e,f) the average integrated optical density (IOD) of the positive cells was measured by ImageJ 1.54p (n = 5). (g–i) HTR7 immunofluorescence staining and HTR7 cell intensities in jejunum and colon were analyzed by ImageJ. (g–j) The expressions of TPH1, HTR7, SERT and β-actin protein were examined in jejunum and colon by Western blot, and relative protein levels were normalized to β-actin (n = 3) (k,l). Each sample was assayed three times. Data are presented as the mean ± SEM. Differences were assessed by ANOVA and denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 indicate significant difference. C: control group; S: restraint stress; S + PCPA: restraint stress + PCPA; S + IWP-2: restraint stress + IWP-2.

Article Snippet: The tissues were incubated overnight at 4 ◦C with the following primary antibodies: rabbit anti-HTR7 antibody (DF13323, Affinity, OH, USA), rabbit anti-LGR5 (DF2816, Affinity, OH, USA), and rabbit anti-β-catenin (66379-1-Ig, Proteintech, Wuhan, China).

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Western Blot, Control

Journal: International journal of molecular sciences

Article Title: Restraint Stress Disrupted Intestinal Homeostasis via 5-HT/HTR7/Wnt/β-Catenin/NF-kB Signaling.

doi: 10.3390/ijms26094021

Figure Lengend Snippet: Figure 4. 5-HT activated Wnt/β-catenin by acting on the HTR7 receptor. The expression of β-catenin and β-actin protein were examined in jejunum (a) and colon (b) by Western blot, and relative protein levels were normalized to β-actin (n = 3). Immunofluorescence staining of β-catenin in Caco2 cells (c) and total and nuclear β-catenin intensities were analyzed by ImageJ (d) (n = 6 hole). 5-HT and SB269970 treated Caco2 cells were used for β-catenin coimmunoprecipitation, input samples from the coimmunoprecipitation assay were analyzed by Western blot (e), and relative protein levels were normalized to β-actin (f) (n = 3). The expressions of TPH1, APC, Axin1, GSK3-β, β-catenin and β-actin protein were examined in jejunum (g) by Western blot, and relative protein levels were normalized to β-actin (h) (n = 3). The expression of β-catenin, TCF3, APC, Axin1, GSK3-β, TPH1, SERT, HTR7 and β-actin protein were examined in colon (i,l) by Western blot, and relative protein levels were normalized to β-actin (j,k,m) (n = 3). Each sample was assayed three times. Data are presented as the mean ± SEM. Differences were assessed by ANOVA and denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 indicate significant difference. C: control group; S: restraint stress; S + PCPA: restraint stress + PCPA; S + IWP-2: restraint stress + IWP-2.

Article Snippet: The tissues were incubated overnight at 4 ◦C with the following primary antibodies: rabbit anti-HTR7 antibody (DF13323, Affinity, OH, USA), rabbit anti-LGR5 (DF2816, Affinity, OH, USA), and rabbit anti-β-catenin (66379-1-Ig, Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Control