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Figure 4.Two-color immunocytochemistry reveals coexpression of Class I OR, TAS2R, TAS1R, and TAAR1 in human blood PMN. (A–C) Coexpression of Class I OR (1:1:1 mix of OR56B4, OR52A4, and OR51B6) and TAS2R43/31. PMN (2474), from n = 7 blood samples, were analyzed. (E–G) Coexpression of Class I OR (1:1:1 mix of OR56B4, OR52A4, and OR51B6) and TAS1R3. PMN (2301), from n = 4 blood samples, were analyzed. (I–K) Coexpression of TAAR1 and TAS1R2. PMN (2377), from n = 4 blood samples, were analyzed. Original scale bars, 5 μm. (D, H, and I) Box-whisker plots display the percentage of receptor-positive cells. Lower and upper blue bars indicate 2nd and 3rd quartiles of data distribution; horizontal lines, median; X, data mean.. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
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Figure 6.G Protein α-gustducin knockdown and lactisole effects on saccharin-induced and TAS1R-mediated PMN migration. (A) RT-qPCR analysis demonstrates relative mRNA expression of Gα-gustducin, Gɣ-13, TRPM5, and Gα-olf in human blood PMN. Data were normalized to an averaged expression of 2 different reference genes as 100%. Shown are means ± sem (n = 7–12). (B) 2-PEA (3 nmole/L) and saccharin (300 µmole/L) stimulated PMN chemotaxis, which was largely abolished by pretreating PMN with PTX (3 µg/ml) or by transfecting them with siRNA against α-gustducin. *P < 0.05 compared with without PTX condition. Data are mean ± sd. (C) Lactisole at 0.3 µmole/L is an antagonist on the saccharin-induced PMN migration. (D) Lactisole itself induces TAS1R3-dependent PMN migration. (B–D) The maximum number of migrated PMN, normalized to a maximum fMLF response (5 nmole/L; n = 4–14).. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
Figure 2.TAS2R and TAS1R gene transcripts are expressed in human blood leukocytes. (A and D) Phylogenetic trees of 25 human TAS2R and 3 TAS1R. (B and E) RT-PCR analysis with gene-specific primers revealed TAS2R and TAS1R mRNA expression with different abundances in different types of blood leukocytes and over n = 28–33 different blood samples. *Cell types with significant, different abundances (ANOVA). (C and F) RT-qPCR demonstrates relative quantitative TAS1R and TAS2R mRNA expression in blood PMN for human receptors (blue) and their cow orthologs (o; red). Data were normalized to an averaged expression of 6 different reference genes, with the lowest values set as 1 (human, TAS2R9 and TAS1R2; cow, TAS2R1 and TAS1R2). (C) Upper scale, Human receptors; lower scale, cow receptors. Shown are means ± sem (n = 15).. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
Figure 1.Class I OR gene transcripts are expressed in human and bovine blood leukocytes. (A) Phylogenetic tree of 55 human Class I OR was calculated by use of the progressive alignment algorithm from CLC Main Workbench (Version 6.8.2; CLCbio/Qiagen, Aarhus, Denmark) software. The tree was created by use of CLC bio’s high-accuracy, neighbor-joining algorithm with 100 bootstrap replicates. (B) RT-PCR analysis with gene-specific primers revealed Class I OR RNA expression with different abundances in different types of blood leukocytes and over n = 10 different blood samples. *Cell types with significant, different abundances (ANOVA). (C) RT-qPCR demonstrates relative quantitative Class I OR mRNA expression in blood PMN for human receptors and their cow orthologs (o). Data were normalized to an averaged expression of 6 different reference genes, with the lowest value [human, OR56B1 (blue); cow, OR51B4 (red)] set as 1. Shown are means ± sem (n = 15).. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
Figure 3.Chemosensory receptor protein expression in subpopulations of human blood leukocytes. Box-whisker plots display the percentage of immunocytochemistry-derived signals of Class I OR (A), TAS1R (B), TAS2R43/31 (C), and TAS2R38 (D) expression in PMN and T and B cells (see also Supplemental Figs. 6–8). Filled bars indicate 2nd and 3rd quartiles of data distribution, with error bars spanning 100% of data. Horizontal lines indicate median; X, data mean. In total, 2749–15,185 PMN, 36,058–76,720 T cells, and 39,496–64,115 B cells from n = 4–11 different blood samples were analyzed. Original magnification, 40×. (For FPR1, see also Supplemental Fig. 9). The efficiency and specificity of TAS1R2 biotin-labeled antibody were confirmed by flow cytometry in PMN under the same experimental conditions as in usual immunocytochemistry. TAS1R2 signals were amplified with streptavidin-Alexa Fluor 633. PMN morphology after cell fixation, and full immunocytochemistry procedure was determined by forward-scatter (FSC; 270 nm) and side-scatter (SSC; 410 nm; E and H) criteria. The intensity of TAS1R2 signals was detected by side-scatter and R1 channel (635 nm; F–J). w/o 1st AB, without primary antibody.. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
Figure 5.TAS1R protein expression and functional relevance of saccharin receptors in human blood PMN. (A–F) Immunocytochemistry and confocal fluorescence microscopy revealed an expression and coexpression of TAS1R2 and TAS1R3 in blood PMN. (A) Blue color indicates nucleus staining; green (B) and red (C) signals represent TAS1R3 and TAS1R2 signals, respectively. (D) Merge of signals from B and C, with overlapping signals appearing in yellow. (E) Overlay of the transmission light picture of the same cell. (F) WB and immunoprecipitation (IP) show the presence of TAS1R2 and TAS1R3 in protein preparations from PMN but not from HeLa, which was used as a negative control. The predicted size of TAS1R2 protein is 95 kDa but may run higher as a result of glycosylation. ‘M, Size marker. (G) Chemotaxis of PMN toward saccharin in a gradient between RPMI and 100 μmole/L saccharin or (H) in RPMI 1640 (negative control) was performed in a 2D CellDirector Opal chamber (Gradientech). Shown are percentage and distance of 40 migrated PMN. Similar results were obtained from 3–6 independent experiments. Transmigration assay (I and J) shows a saccharin-induced TAS1R2-, TAS1R3-, TAS2R31-, and TAS2R43- but not TAS1R1-dependent PMN migration. PMN migration, Normalized to a maximum fMLF response (5 nmole/L; n = 8–12); non-siRNA represents a negative knockdown control; there, none of the receptors are silenced. Data are mean ± sd.. From: Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
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