Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: Classification of hub genes (A–C) . The difference in HTRA1 ( p = 2.222e-08), P2RY6 ( p = 1.335e-06), and RCAN1 ( p = 0.001) levels could be observed in OA compared with control groups. HTRA1, HtrA serine peptidase 1; P2RY6, pyrimidinergic receptor P2Y6; RCAN1, regulator of calcineurin 1.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques:

Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: GSVA. Pathways enriched by (A) HTRA1; (B) P2RY6; and (C) RCAN1. A positive association is symbolized by a blue band, and a negative association is symbolized by a green band.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques:

Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: By adopting RT-qPCR, the above 3 hub genes were discovered to be differentially expressed in the OA group in relative to the control group. (A) HTRA1, (D) P2RY6, and (G) RCAN1. GAPDH expression was taken as standard for all samples. Means ±SEM ( n = 9) are used to represent values, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, OA, osteoarthritis. (B,C,E,F,H,I) Quantitative analysis and Western blot assay exhibiting HTRA1, P2RY6, RCAN1 protein levels, and corresponding NCs, with GAPDH being the endogenous reference. In addition, data are indicated by means ±SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: MSU binding to P2Y6 receptor induces CXCL8 and IL-1β production, and PLAG modulates CXCL8 expression but not IL-1β expression. (A) Time effect of PLAG on the mRNA expression of MSU-induced CXCL8 and IL-1β. THP-1 cells were pre-treated with PLAG (100 μg/ml) for 1 h and then were treated with MSU crystals (200 μg/ml). (B,C) Quantification of (A) . (D) Dose effect of PLAG on the mRNA expression of MSU-induced CXCL8 and IL-1β.THP-1 cells were pre-treated with different dose of PLAG (1, 10, or 100 μg/ml) and then were treated with MSU crystals (200 μg/ml). (E,F) Quantification of (D) . GAPDH was used as a control (G,H) Secreted CXCL8 and IL-1β were measured using ELISA in the supernatants of THP-1 cells treated with different doses of MSU crystals (50, 200, or 1,000 μg/ml). (I,J) Secreted CXCL8 and IL-1β in the supernatant of THP-1 cells pre-treated with different doses of PLAG (0.1, 1, 10, or 100 μg/ml) for 1h and then treated with MSU crystals (200 μg/ml) for 24 h were measured using ELISA. (K,L) THP-1 cells were pre-treated with MRS2578 (0.5, 1, or 2 μM) and treated with MSU crystals (200 μg/ml). Secreted CXCL8 and IL-1β in the supernatants of THP-1 cells were measured using ELISA. (M,N) THP-1 cells were transfected with control or P2Y6 receptor siRNA (50 nM) for 24 h. P2Y6-knockdown THP-1 cells were treated with MSU crystals (200 μg/ml) for 24 h, and secreted CXCL8 and IL-1β in the supernatants of THP-1 cells were measured using ELISA. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The specific short interfering RNAs (siRNAs) of human P2Y6 receptor (sc-42584), TRIF (sc-106845), MyD88 (sc-35986), and control siRNA (sc-37007), were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Expressing, Control, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: Accelerated intracellular trafficking of MSU-loaded P2Y6 in PLAG-treated cells. (A) Analysis of cell surface P2Y6 receptor using confocal microscopy. THP-1 cells were pre-treated with PLAG (100 μg/ml) for 1 h and then were treated with MSU (200 μg/ml). The cells were harvested and were fixed with 4% paraformaldehyde. The membrane-localized receptor was stained with anti-P2Y6 receptor antibody without permeabilization. (B) The result of (A) was also analyzed by flow cytometry. (C) Lysosomal activity was measured using lyso-ID and analyzed using confocal microscopy. (D) The result of (C) was also analyzed by flow cytometry. (E) Co-localization of P2Y6 receptor and Rab5 was examined using confocal microscopy. (F) Graphs show fluorescence intensity profiles calculated on the white arrows of the confocal images in (E) using ZEN program. Data are presented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The specific short interfering RNAs (siRNAs) of human P2Y6 receptor (sc-42584), TRIF (sc-106845), MyD88 (sc-35986), and control siRNA (sc-37007), were purchased from Santa Cruz Biotechnology.

Techniques: Confocal Microscopy, Membrane, Staining, Flow Cytometry, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: PLAG phosphorylates GRK2 and P2Y6 receptor to form a receptor endocytosis-related protein complex. (A) Effect of PLAG on phosphorylation of GRK2. THP-1 cells were treated with 100 μg/ml PLAG for 0, 7, 15, 30, and 60 min. Whole lysates were used to analyze phosphorylated GRK2 and total protein of GRK2. (B) Quantification of (A) . (C) Effect of PLAG on phosphorylation of P2Y6 receptor. Cell lysates were immunoblotted with phospho-threonine antibody after immunoprecipitation with the P2Y6 receptor antibody. (D) Quantification of (C) . (E) THP-1 cells were incubated with 100 μg/ml PLAG for 1 h and then were stimulated with 200 μg/ml MSU crystals for 0, 7, 15, 30, and 60 min. Whole lysates were used to analyze phosphorylated GRK2 and total protein of GRK2. (F) Quantification of (E) . (G) Cell lysates were immunoblotted with phospho-threonine antibody after immunoprecipitation with the P2Y6 receptor antibody. (H) Quantification of (G) . (I) Cell lysates were immunoblotted with α-arrestin, β-arrestin2, or clathrin antibodies after immunoprecipitation with the P2Y6 receptor antibody. (J,K,L) Quantification of (I) . (M) Schematic diagram of endocytosis-related protein complex formation by PLAG treatment for 1 h. One hour after PLAG treatment, GRK2 and P2Y6R were phosphorylated, and endocytosis-related proteins such as α-arrestin, β-arrestin, and clathrin were complexed with P2Y6R. (N) Schematic diagram of P2Y6 receptor endocytosis and duration of intracellular trafficking in MSU-treated THP-1 cells. PLAG, using a preformed endocytosis-related complex, promotes the initiation of intracellular trafficking of P2Y6R and rapidly brings it back to the cell surface when MSU is treated. Therefore, PLAG shortens the duration of P2Y6R stays in the cytoplasm.

Article Snippet: The specific short interfering RNAs (siRNAs) of human P2Y6 receptor (sc-42584), TRIF (sc-106845), MyD88 (sc-35986), and control siRNA (sc-37007), were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Immunoprecipitation, Incubation

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: The reduction of CXCL8 expression by PLAG is dependent on TRIF/IRF3 signaling. (A) THP-1 cells were transfected with TRIF and MyD88 siRNA. After 24 h, the cells were incubated with 100 μg/ml PLAG or DMSO for 1 h. The cells were then treated with 200 μg/ml of MSU crystals for 24 h. The culture supernatants were harvested and assayed using ELISA to check the secreted levels of CXCL8. (B) THP-1 cells were incubated with 100 μg/ml PLAG or DMSO for 1 h and were stimulated with 200 μg/ml MSU crystals for 7, 15, 30, and 60 min. Whole lysates were used to analyze the phosphorylation of TRAM. TRAM was used as the loading control. (C) Quantification of (B) . (D) TRIF signaling during trafficking was evaluated in THP-1 cells. Cells were incubated with 100 μg/ml PLAG or DMSO for 1 h and were stimulated with 200 μg/ml of MSU crystals for 7, 15, 30, and 60 min. Cell lysates were immunoblotted with TRIF and MyD88 antibodies after immunoprecipitation with the P2Y6 receptor antibody. (E,F) Quantification of (D) . (G) Whole lysates were used to analyze the phosphorylation of IRF-3 or IκBα degradation. β-Actin was used as a loading control. (H,I) Quantification of (G) . (J) A schematic diagram showing the mechanism of action of PLAG. PLAG promotes MSU-induced P2Y6 receptor intracellular trafficking (1) and reduces the duration of endocytosis-dependent signaling (2). Consequently, PLAG reduces MSU-induced CXCL8 production (3) and prevents excessive neutrophil recruitment into MSU-accumulated tissue (4). Data are presented as mean ± SD. *** p < 0.001, compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The specific short interfering RNAs (siRNAs) of human P2Y6 receptor (sc-42584), TRIF (sc-106845), MyD88 (sc-35986), and control siRNA (sc-37007), were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: Specificity of PLAG in accelerated P2Y6 endocytosis and the modulation of CXCL8 expression. (A) Chemical structure of PLAG and PLH. (B) Advanced endocytosis by PLAG was compared with that by PLH. Cells were incubated with 100 μg/ml PLAG, PLH, or DMSO for 1 h and then stimulated with 200 μg/ml of MSU crystals. The cells were harvested over time were fixed using 4% paraformaldehyde. P2Y6 receptor expressed on the membrane was stained with the P2Y6 receptor antibody without permeabilization and was analyzed using confocal microscopy. (C) The result of B was also confirmed by flow cytometry. (D,E) PLAG was compared with PLH to check the secretion levels of CXCL8 and IL-1β. The cells were incubated with 100 μg/ml PLAG, PLH, or DMSO for 1 h and were treated with 200 μg/ml MSU crystals for 24 h. The culture supernatants were harvested and used for ELISA. Data are presented as mean ± SD. *** p < 0.001, compared to the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, statistical difference between the MSU-treated group and MSU + PLAG treated group; $p < 0.05, statistical difference between the MSU-treated group and MSU + PLH treated group; & p < 0.05, statistical difference between the MSU + PLAG-treated group and MSU + PLH-treated group.

Article Snippet: The specific short interfering RNAs (siRNAs) of human P2Y6 receptor (sc-42584), TRIF (sc-106845), MyD88 (sc-35986), and control siRNA (sc-37007), were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Incubation, Membrane, Staining, Confocal Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: MSU binding to P2Y6 receptor induces CXCL8 and IL-1β production, and PLAG modulates CXCL8 expression but not IL-1β expression. (A) Time effect of PLAG on the mRNA expression of MSU-induced CXCL8 and IL-1β. THP-1 cells were pre-treated with PLAG (100 μg/ml) for 1 h and then were treated with MSU crystals (200 μg/ml). (B,C) Quantification of (A) . (D) Dose effect of PLAG on the mRNA expression of MSU-induced CXCL8 and IL-1β.THP-1 cells were pre-treated with different dose of PLAG (1, 10, or 100 μg/ml) and then were treated with MSU crystals (200 μg/ml). (E,F) Quantification of (D) . GAPDH was used as a control (G,H) Secreted CXCL8 and IL-1β were measured using ELISA in the supernatants of THP-1 cells treated with different doses of MSU crystals (50, 200, or 1,000 μg/ml). (I,J) Secreted CXCL8 and IL-1β in the supernatant of THP-1 cells pre-treated with different doses of PLAG (0.1, 1, 10, or 100 μg/ml) for 1h and then treated with MSU crystals (200 μg/ml) for 24 h were measured using ELISA. (K,L) THP-1 cells were pre-treated with MRS2578 (0.5, 1, or 2 μM) and treated with MSU crystals (200 μg/ml). Secreted CXCL8 and IL-1β in the supernatants of THP-1 cells were measured using ELISA. (M,N) THP-1 cells were transfected with control or P2Y6 receptor siRNA (50 nM) for 24 h. P2Y6-knockdown THP-1 cells were treated with MSU crystals (200 μg/ml) for 24 h, and secreted CXCL8 and IL-1β in the supernatants of THP-1 cells were measured using ELISA. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 (Calbiochem) for 1 h. It was then incubated with anti-phospho-GRK2 (Thermo Fisher Scientific Inc.), GRK2 (Santa Cruz Biotechnology), phospho-threonine (Cell Signaling Technology), α-arrestin (Cell Signaling Technology), clathrin (Abcam), β-arrestin2 (Santa Cruz Biotechnology), P2Y6 receptor (Alomone Labs), phospho-TRAM (Mybiosource, San Diego, USA), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), TRIF (Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Binding Assay, Expressing, Control, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: Accelerated intracellular trafficking of MSU-loaded P2Y6 in PLAG-treated cells. (A) Analysis of cell surface P2Y6 receptor using confocal microscopy. THP-1 cells were pre-treated with PLAG (100 μg/ml) for 1 h and then were treated with MSU (200 μg/ml). The cells were harvested and were fixed with 4% paraformaldehyde. The membrane-localized receptor was stained with anti-P2Y6 receptor antibody without permeabilization. (B) The result of (A) was also analyzed by flow cytometry. (C) Lysosomal activity was measured using lyso-ID and analyzed using confocal microscopy. (D) The result of (C) was also analyzed by flow cytometry. (E) Co-localization of P2Y6 receptor and Rab5 was examined using confocal microscopy. (F) Graphs show fluorescence intensity profiles calculated on the white arrows of the confocal images in (E) using ZEN program. Data are presented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 (Calbiochem) for 1 h. It was then incubated with anti-phospho-GRK2 (Thermo Fisher Scientific Inc.), GRK2 (Santa Cruz Biotechnology), phospho-threonine (Cell Signaling Technology), α-arrestin (Cell Signaling Technology), clathrin (Abcam), β-arrestin2 (Santa Cruz Biotechnology), P2Y6 receptor (Alomone Labs), phospho-TRAM (Mybiosource, San Diego, USA), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), TRIF (Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Confocal Microscopy, Membrane, Staining, Flow Cytometry, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: PLAG phosphorylates GRK2 and P2Y6 receptor to form a receptor endocytosis-related protein complex. (A) Effect of PLAG on phosphorylation of GRK2. THP-1 cells were treated with 100 μg/ml PLAG for 0, 7, 15, 30, and 60 min. Whole lysates were used to analyze phosphorylated GRK2 and total protein of GRK2. (B) Quantification of (A) . (C) Effect of PLAG on phosphorylation of P2Y6 receptor. Cell lysates were immunoblotted with phospho-threonine antibody after immunoprecipitation with the P2Y6 receptor antibody. (D) Quantification of (C) . (E) THP-1 cells were incubated with 100 μg/ml PLAG for 1 h and then were stimulated with 200 μg/ml MSU crystals for 0, 7, 15, 30, and 60 min. Whole lysates were used to analyze phosphorylated GRK2 and total protein of GRK2. (F) Quantification of (E) . (G) Cell lysates were immunoblotted with phospho-threonine antibody after immunoprecipitation with the P2Y6 receptor antibody. (H) Quantification of (G) . (I) Cell lysates were immunoblotted with α-arrestin, β-arrestin2, or clathrin antibodies after immunoprecipitation with the P2Y6 receptor antibody. (J,K,L) Quantification of (I) . (M) Schematic diagram of endocytosis-related protein complex formation by PLAG treatment for 1 h. One hour after PLAG treatment, GRK2 and P2Y6R were phosphorylated, and endocytosis-related proteins such as α-arrestin, β-arrestin, and clathrin were complexed with P2Y6R. (N) Schematic diagram of P2Y6 receptor endocytosis and duration of intracellular trafficking in MSU-treated THP-1 cells. PLAG, using a preformed endocytosis-related complex, promotes the initiation of intracellular trafficking of P2Y6R and rapidly brings it back to the cell surface when MSU is treated. Therefore, PLAG shortens the duration of P2Y6R stays in the cytoplasm.

Article Snippet: The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 (Calbiochem) for 1 h. It was then incubated with anti-phospho-GRK2 (Thermo Fisher Scientific Inc.), GRK2 (Santa Cruz Biotechnology), phospho-threonine (Cell Signaling Technology), α-arrestin (Cell Signaling Technology), clathrin (Abcam), β-arrestin2 (Santa Cruz Biotechnology), P2Y6 receptor (Alomone Labs), phospho-TRAM (Mybiosource, San Diego, USA), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), TRIF (Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Phospho-proteomics, Immunoprecipitation, Incubation

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: The reduction of CXCL8 expression by PLAG is dependent on TRIF/IRF3 signaling. (A) THP-1 cells were transfected with TRIF and MyD88 siRNA. After 24 h, the cells were incubated with 100 μg/ml PLAG or DMSO for 1 h. The cells were then treated with 200 μg/ml of MSU crystals for 24 h. The culture supernatants were harvested and assayed using ELISA to check the secreted levels of CXCL8. (B) THP-1 cells were incubated with 100 μg/ml PLAG or DMSO for 1 h and were stimulated with 200 μg/ml MSU crystals for 7, 15, 30, and 60 min. Whole lysates were used to analyze the phosphorylation of TRAM. TRAM was used as the loading control. (C) Quantification of (B) . (D) TRIF signaling during trafficking was evaluated in THP-1 cells. Cells were incubated with 100 μg/ml PLAG or DMSO for 1 h and were stimulated with 200 μg/ml of MSU crystals for 7, 15, 30, and 60 min. Cell lysates were immunoblotted with TRIF and MyD88 antibodies after immunoprecipitation with the P2Y6 receptor antibody. (E,F) Quantification of (D) . (G) Whole lysates were used to analyze the phosphorylation of IRF-3 or IκBα degradation. β-Actin was used as a loading control. (H,I) Quantification of (G) . (J) A schematic diagram showing the mechanism of action of PLAG. PLAG promotes MSU-induced P2Y6 receptor intracellular trafficking (1) and reduces the duration of endocytosis-dependent signaling (2). Consequently, PLAG reduces MSU-induced CXCL8 production (3) and prevents excessive neutrophil recruitment into MSU-accumulated tissue (4). Data are presented as mean ± SD. *** p < 0.001, compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the MSU-treated group.

Article Snippet: The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 (Calbiochem) for 1 h. It was then incubated with anti-phospho-GRK2 (Thermo Fisher Scientific Inc.), GRK2 (Santa Cruz Biotechnology), phospho-threonine (Cell Signaling Technology), α-arrestin (Cell Signaling Technology), clathrin (Abcam), β-arrestin2 (Santa Cruz Biotechnology), P2Y6 receptor (Alomone Labs), phospho-TRAM (Mybiosource, San Diego, USA), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), TRIF (Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Expressing, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice

doi: 10.3389/fimmu.2020.00710

Figure Lengend Snippet: Specificity of PLAG in accelerated P2Y6 endocytosis and the modulation of CXCL8 expression. (A) Chemical structure of PLAG and PLH. (B) Advanced endocytosis by PLAG was compared with that by PLH. Cells were incubated with 100 μg/ml PLAG, PLH, or DMSO for 1 h and then stimulated with 200 μg/ml of MSU crystals. The cells were harvested over time were fixed using 4% paraformaldehyde. P2Y6 receptor expressed on the membrane was stained with the P2Y6 receptor antibody without permeabilization and was analyzed using confocal microscopy. (C) The result of B was also confirmed by flow cytometry. (D,E) PLAG was compared with PLH to check the secretion levels of CXCL8 and IL-1β. The cells were incubated with 100 μg/ml PLAG, PLH, or DMSO for 1 h and were treated with 200 μg/ml MSU crystals for 24 h. The culture supernatants were harvested and used for ELISA. Data are presented as mean ± SD. *** p < 0.001, compared to the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, statistical difference between the MSU-treated group and MSU + PLAG treated group; $p < 0.05, statistical difference between the MSU-treated group and MSU + PLH treated group; & p < 0.05, statistical difference between the MSU + PLAG-treated group and MSU + PLH-treated group.

Article Snippet: The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 (Calbiochem) for 1 h. It was then incubated with anti-phospho-GRK2 (Thermo Fisher Scientific Inc.), GRK2 (Santa Cruz Biotechnology), phospho-threonine (Cell Signaling Technology), α-arrestin (Cell Signaling Technology), clathrin (Abcam), β-arrestin2 (Santa Cruz Biotechnology), P2Y6 receptor (Alomone Labs), phospho-TRAM (Mybiosource, San Diego, USA), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), TRIF (Cell Signaling Technology), MyD88 (D80F5, Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight.

Techniques: Expressing, Incubation, Membrane, Staining, Confocal Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Primary and secondary antibodies used in immunohistochemistry experiments

Article Snippet: Probes Open in a separate window Primary and secondary antibodies used in immunohistochemistry experiments To test the specificity of the antibody for P2Y 6 receptors (APR-011), some sections were processed with the primary antibody pre-adsorbed with a control antigen corresponding to the amino acid sequence 311–328 of the rat P2Y 6 receptor ( {"type":"entrez-protein","attrs":{"text":"Q63371","term_id":"2495019","term_text":"Q63371"}} Q63371 , Alomone Labs, Jerusalem, Israel).

Techniques: Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis *

doi: 10.1074/jbc.M116.760322

Figure Lengend Snippet: The P2Y receptors and signaling pathways involved in the UTP-induced HAS2 up-regulation. HaCaT cells were transfected with control and P2Y2-specific siRNAs (A and B). A, 2 days after the transfection the samples were collected to test for the efficiency of the siRNAs (n = 4), or B, subjected to 100 μm UTP for 2 h prior to collecting the samples for HAS2 qRT-PCR (n = 5). C and D, cells were subjected to MRS2578 (a selective antagonist of P2Y6, 20 μm) for 30 min, and E and F, PTX (100 ng/ml) for 17 h prior to the addition of 100 μm UDP (C and E) and UTP (D and F) for 2 h before mRNA assays. Statistical significances of the differences between the groups were tested using one group t test in A (##, p < 0.01). Mixed model ANOVA was used for comparisons between the different treatments (indicated by **, p < 0.01; ***, p < 0.001) and comparisons of treatments to controls (set to 1) was tested by pnorm (indicated by #, p < 0.05; ##, p < 0.01; ###, p < 0.001) in B–F.

Article Snippet: Equal amounts of these solvents were added to the control cultures. siRNA Treatments The control siRNAs were obtained from Eurogentec (Liege, Belgium), P2Y 2 -targeted siRNAs were from Thermo Fisher (Waltham, MA), and P2Y 6 -targeted siRNAs from Origene (Rockville, MD).

Techniques: Transfection, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis *

doi: 10.1074/jbc.M116.760322

Figure Lengend Snippet: Schematic representation of the signaling pathways involved in the UTP-induced HAS2 up-regulation. Extracellular UTP and its breakdown product UDP activate the P2Y receptors that increase HAS2 expression via the indicated signaling steps. Those steps positively verified in the current study are marked green, whereas the pathways excluded are indicated by an “x.”

Article Snippet: Equal amounts of these solvents were added to the control cultures. siRNA Treatments The control siRNAs were obtained from Eurogentec (Liege, Belgium), P2Y 2 -targeted siRNAs were from Thermo Fisher (Waltham, MA), and P2Y 6 -targeted siRNAs from Origene (Rockville, MD).

Techniques: Expressing