Journal: The Journal of Biological Chemistry

Article Title: Neuroendocrine Signaling Via the Serotonin Transporter Regulates Clearance of Apoptotic Cells *

doi: 10.1074/jbc.M113.482299

Figure Lengend Snippet: 5-HT receptor 2a, 2b, and the 5-HT transporter are expressed in mouse peritoneal macrophages. A, real-time PCR was performed to measure the expression by mRNA. 5-HT receptor 2a (5HTR2a), 5-HT receptor 2b (5HTR2b), and the 5-HTT were expressed in TG-elicited peritoneal macrophages, but 5-HT receptor 2c (5HTR2c) was not detected. Data represent the mean ± S.E. for n = 3 per group. B–D, Western blotting is shown for two representative peritoneal macrophage lysates (PM1 and PM2) evaluating expression of 5-HT receptors and transporter. B, HTR2a (n = 4); C, HTR2b (n = 10); and D, 5-HTT (n = 8) were all detected in mouse peritoneal macrophages.

Article Snippet: Inventoried TaqMan Gene Expression Assays (Applied Biosystems) were used to measure mRNA expression levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, assay ID Mm03302249_g1), HTR2a (htr2a, assay ID Mm00555764_m1), HTR2b (htr2b, assay ID Mm00434123_m1), and 5-HT transporter (SLC6A4, assay ID Mm00439391_m1).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Neuroendocrine Signaling Via the Serotonin Transporter Regulates Clearance of Apoptotic Cells *

doi: 10.1074/jbc.M113.482299

Figure Lengend Snippet: Effect of 5-HTT deficiency on 5-HT uptake, efferocytosis, Rho kinase activity, and peritoneal inflammation. A, function of the 5-HTT in TG-elicited peritoneal macrophages was investigated using a neuroendocrine uptake assay, where a tracer becomes fluorescent upon internalization. The tracer can be internalized by the 5-HT, dopamine, or the norepinephrine transporters, so specificity depends on genetic deletion of a given transporter. TG-elicited peritoneal macrophages were isolated from 5-HTT KO (gray circles) and wild-type control mice (black circles), and uptake of the fluorescent tracer was assessed and expressed as relative fluorescent units (RFU). Lines indicate paired experiments. p = 0.016, wild-type versus 5-HTT KO mice (paired t test). B, TG-elicited peritoneal macrophages from wild-type mice (black columns) and 5-HTT KO mice (gray columns) were treated with media alone or media containing 5-HT (1 μm) for 24 h. Apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO2 at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. 5-HT decreased efferocytosis in wild-type macrophages. Data represent the mean ± S.E. for n = 8 per group. Two-way ANOVA, p < 0.001 for interaction, and p < 0.01 for treatment effect (media versus 5-HT treatment). *, Tukey-Kramer post hoc analysis, p < 0.001 for wild-type 5-HT treatment versus wild-type media control; p < 0.01 for wild-type 5-HT treatment versus 5-HTT KO 5-HT treatment; p < 0.05 for wild-type 5-HT treatment versus 5-HTT KO media control. C, murine TG-elicited peritoneal macrophages from wild-type (black columns) and 5-HTT KO mice (gray columns) were stimulated with 5-HT (1 μm) for the indicated times. Densitometry was used to determine the ratio of phosphomyosin phosphatase 1 (pMypt1) to total Mypt1 by arbitrary units. 5-HT increased phosphorylation of Mypt1 at Thr-696 in wild-type but not in 5-HTT KO macrophages. Data represent the mean ± S.E. for n ≤ 13 per group. Two-way ANOVA, p = 0.03 for interaction, and p < 0.01 for genotype effect (wild-type versus 5-HTT KO). *, Tukey-Kramer post hoc analysis p < 0.03 for wild-type media control versus wild-type 5-HT treatment for 5 min; **, p < 0.01 for wild-type media control versus wild-type 5-HT treatment for 20 min; †, p < 0.03 for wild-type 5-HT treatment for 20 min versus 5-HTT KO 5-HT treatment for 20 min. D, 5-HTT KO (gray circles) and wild-type (black circles) mice were injected with TG. Four days later peritoneal lavage was performed and total cell counts and differentials were taken. Neutrophils were decreased in 5-HTT KO mice compared with wild-type mice. Data represent the mean ± S.E. for n = 8–10 mice per group. p = 0.036, wild-type versus 5-HTT KO mice (Mann-Whitney test).

Article Snippet: Inventoried TaqMan Gene Expression Assays (Applied Biosystems) were used to measure mRNA expression levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, assay ID Mm03302249_g1), HTR2a (htr2a, assay ID Mm00555764_m1), HTR2b (htr2b, assay ID Mm00434123_m1), and 5-HT transporter (SLC6A4, assay ID Mm00439391_m1).

Techniques: Activity Assay, Isolation, Control, Cell Culture, Phospho-proteomics, Injection, MANN-WHITNEY