tmem16a  (Alomone Labs)


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    Structured Review

    Alomone Labs tmem16a
    Effect of dextran sulfate sodium (DSS)-colitis on Cl − channel protein expression in murine colon. A : representative images of protein expression of the different characterized Cl − channels from epithelial lysates. BEST2, bestrophin-2. B : transmembrane protein 16A <t>(TMEM16A)</t> protein expression normalized to β-actin for quantification of control and DSS-colitis groups. TMEM16A expression (closed bar) is significantly decreased in DSS-colitis mice as compared with control. C and D : summarized data of Western blot quantification of BEST2 and CFTR normalized to β-actin in control and DSS-colitis mice. Neither protein was significantly altered from the control cohort. Bar graphs of summarized data represent means ± SE of 5 different animals from each respective group. * P
    Tmem16a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Pathogenic Roles of Ion Channels and Transporters: Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl− secretion in murine colon by downregulating TMEM16A"

    Article Title: Pathogenic Roles of Ion Channels and Transporters: Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl− secretion in murine colon by downregulating TMEM16A

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00328.2017

    Effect of dextran sulfate sodium (DSS)-colitis on Cl − channel protein expression in murine colon. A : representative images of protein expression of the different characterized Cl − channels from epithelial lysates. BEST2, bestrophin-2. B : transmembrane protein 16A (TMEM16A) protein expression normalized to β-actin for quantification of control and DSS-colitis groups. TMEM16A expression (closed bar) is significantly decreased in DSS-colitis mice as compared with control. C and D : summarized data of Western blot quantification of BEST2 and CFTR normalized to β-actin in control and DSS-colitis mice. Neither protein was significantly altered from the control cohort. Bar graphs of summarized data represent means ± SE of 5 different animals from each respective group. * P
    Figure Legend Snippet: Effect of dextran sulfate sodium (DSS)-colitis on Cl − channel protein expression in murine colon. A : representative images of protein expression of the different characterized Cl − channels from epithelial lysates. BEST2, bestrophin-2. B : transmembrane protein 16A (TMEM16A) protein expression normalized to β-actin for quantification of control and DSS-colitis groups. TMEM16A expression (closed bar) is significantly decreased in DSS-colitis mice as compared with control. C and D : summarized data of Western blot quantification of BEST2 and CFTR normalized to β-actin in control and DSS-colitis mice. Neither protein was significantly altered from the control cohort. Bar graphs of summarized data represent means ± SE of 5 different animals from each respective group. * P

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Immunohistochemistry of transmembrane protein 16A (TMEM16A) in colonic tissue sections. A : immunohistochemistry of TMEM16A in control tissue demonstrates pronounced expression and localization of the protein to surface epithelium and upper crypts ( top , left ). B. DSS-colitis tissue stained for TMEM16A exhibits less staining in epithelial cells and a more diffuse signal in the lamina propria ( bottom , left ). C and D : both cohorts of tissue were run in parallel with a control peptide specific for the anti-TMEM16A antibody. Both images demonstrate a lack of staining when coincubated with control peptide. Images were captured at ×20 magnification on the AxioImager (Carl Zeiss, Germany).
    Figure Legend Snippet: Immunohistochemistry of transmembrane protein 16A (TMEM16A) in colonic tissue sections. A : immunohistochemistry of TMEM16A in control tissue demonstrates pronounced expression and localization of the protein to surface epithelium and upper crypts ( top , left ). B. DSS-colitis tissue stained for TMEM16A exhibits less staining in epithelial cells and a more diffuse signal in the lamina propria ( bottom , left ). C and D : both cohorts of tissue were run in parallel with a control peptide specific for the anti-TMEM16A antibody. Both images demonstrate a lack of staining when coincubated with control peptide. Images were captured at ×20 magnification on the AxioImager (Carl Zeiss, Germany).

    Techniques Used: Immunohistochemistry, Expressing, Staining

    Effect of dextran sulfate sodium (DSS)-colitis on transcript abundance of Cl − channels in murine colon. Transcript abundance was determined for transmembrane protein 16A ( Tmem16a ; A ), bestrophin-2 ( Best2 ; B ), and Cftr ( C ) in control (open bars) or DSS-colitis mice (closed bars). Bar graphs of summarized data represent means ± SE of 5 different animals normalized to an endogenous control, Actb . * P
    Figure Legend Snippet: Effect of dextran sulfate sodium (DSS)-colitis on transcript abundance of Cl − channels in murine colon. Transcript abundance was determined for transmembrane protein 16A ( Tmem16a ; A ), bestrophin-2 ( Best2 ; B ), and Cftr ( C ) in control (open bars) or DSS-colitis mice (closed bars). Bar graphs of summarized data represent means ± SE of 5 different animals normalized to an endogenous control, Actb . * P

    Techniques Used: Mouse Assay

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    Alomone Labs anti at1r antibody
    Schematic diagram of signal transduction pathways involved in Ang II-induced endothelial hyperpermeability via <t>AT1R/RAGE/mDia1/Src/β-catenin/VE-cadherin.</t> a As Ang II binds to and stimulates AT1R, the expression and secretion of HMGB1 can be increased by NF-κB activation. NF-κB-mediated expression of proinflammatory molecules, including RAGE itself, can occur. HMGB1 binds to RAGE, which can induce RAGE-mediated activation of Src/β-catenin/VE-cadherin via mDia1. b Blockade of RAGE activation by sRAGE attenuates the Ang II-induced increase in endothelial hyperpermeability by inhibiting RAGE-mediated signaling pathways
    Anti At1r Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs p2y6 receptors
    RT-PCR and sequence analysis of porcine P2Y2 , P2Y4 and <t>P2Y6</t> receptors
    P2y6 Receptors, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic diagram of signal transduction pathways involved in Ang II-induced endothelial hyperpermeability via AT1R/RAGE/mDia1/Src/β-catenin/VE-cadherin. a As Ang II binds to and stimulates AT1R, the expression and secretion of HMGB1 can be increased by NF-κB activation. NF-κB-mediated expression of proinflammatory molecules, including RAGE itself, can occur. HMGB1 binds to RAGE, which can induce RAGE-mediated activation of Src/β-catenin/VE-cadherin via mDia1. b Blockade of RAGE activation by sRAGE attenuates the Ang II-induced increase in endothelial hyperpermeability by inhibiting RAGE-mediated signaling pathways

    Journal: Experimental & Molecular Medicine

    Article Title: Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE

    doi: 10.1038/s12276-019-0312-5

    Figure Lengend Snippet: Schematic diagram of signal transduction pathways involved in Ang II-induced endothelial hyperpermeability via AT1R/RAGE/mDia1/Src/β-catenin/VE-cadherin. a As Ang II binds to and stimulates AT1R, the expression and secretion of HMGB1 can be increased by NF-κB activation. NF-κB-mediated expression of proinflammatory molecules, including RAGE itself, can occur. HMGB1 binds to RAGE, which can induce RAGE-mediated activation of Src/β-catenin/VE-cadherin via mDia1. b Blockade of RAGE activation by sRAGE attenuates the Ang II-induced increase in endothelial hyperpermeability by inhibiting RAGE-mediated signaling pathways

    Article Snippet: Anti-AT1R antibody (AAR-011) was purchased from Alomone Labs (Hadassah Ein Kerem, Israel).

    Techniques: Transduction, Expressing, Activation Assay

    Importance of the AT1R-RAGE axis in Ang II-induced endothelial hyperpermeability in HUVECs. a Changes in the phosphorylation of VE-cadherin (Y731), Src (Tyr416), and β-catenin (Ser552) in HUVECs following transfection with RAGE siRNA and treatment with Ang II. Expression values were normalized to those of VE-cadherin, Src, and β-catenin ( n = 4 for each lane). b Immunocytochemistry of VE-cadherin (green) and DAPI (nuclei, blue), as examined under a confocal microscope (white scale bars: 50 μm in merged images and 20 μm in magnified images; ×400 magnification). The main images were selected from representative regions. c HUVECs transfected with siRNA targeting RAGE or with scrambled sequences were incubated for 72 h and were then stimulated with Ang II for 6 h. TEER was measured every 2 h. *** p

    Journal: Experimental & Molecular Medicine

    Article Title: Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE

    doi: 10.1038/s12276-019-0312-5

    Figure Lengend Snippet: Importance of the AT1R-RAGE axis in Ang II-induced endothelial hyperpermeability in HUVECs. a Changes in the phosphorylation of VE-cadherin (Y731), Src (Tyr416), and β-catenin (Ser552) in HUVECs following transfection with RAGE siRNA and treatment with Ang II. Expression values were normalized to those of VE-cadherin, Src, and β-catenin ( n = 4 for each lane). b Immunocytochemistry of VE-cadherin (green) and DAPI (nuclei, blue), as examined under a confocal microscope (white scale bars: 50 μm in merged images and 20 μm in magnified images; ×400 magnification). The main images were selected from representative regions. c HUVECs transfected with siRNA targeting RAGE or with scrambled sequences were incubated for 72 h and were then stimulated with Ang II for 6 h. TEER was measured every 2 h. *** p

    Article Snippet: Anti-AT1R antibody (AAR-011) was purchased from Alomone Labs (Hadassah Ein Kerem, Israel).

    Techniques: Transfection, Expressing, Immunocytochemistry, Microscopy, Incubation

    HMGB1 is an important mediator of AT1R-RAGE signaling. HUVECs were treated with Ang II in the absence or presence of losartan for 4 h. a HMGB1 release in supernatants was measured by ELISA and western blotting ( n = 4 for each lane). b , c HUVECs were incubated with Ang II in the presence or absence of anti-HMGB1-neutralizing antibody (50 ng/ml) for 4 h and analyzed by western blotting. The relative values of AT1R, RAGE, and mDia1 expression were normalized to that of GAPDH ( n = 4 for each lane). The relative values of phospho-VE-cadherin expression were normalized to that of VE-cadherin ( n = 3 for each lane). The values are presented as the means ± SEMs. * p

    Journal: Experimental & Molecular Medicine

    Article Title: Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE

    doi: 10.1038/s12276-019-0312-5

    Figure Lengend Snippet: HMGB1 is an important mediator of AT1R-RAGE signaling. HUVECs were treated with Ang II in the absence or presence of losartan for 4 h. a HMGB1 release in supernatants was measured by ELISA and western blotting ( n = 4 for each lane). b , c HUVECs were incubated with Ang II in the presence or absence of anti-HMGB1-neutralizing antibody (50 ng/ml) for 4 h and analyzed by western blotting. The relative values of AT1R, RAGE, and mDia1 expression were normalized to that of GAPDH ( n = 4 for each lane). The relative values of phospho-VE-cadherin expression were normalized to that of VE-cadherin ( n = 3 for each lane). The values are presented as the means ± SEMs. * p

    Article Snippet: Anti-AT1R antibody (AAR-011) was purchased from Alomone Labs (Hadassah Ein Kerem, Israel).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Expressing

    RAGE regulates Ang II-induced endothelial hyperpermeability via mDia1. a HUVECs were transfected with mDia1 siRNA and cultured in the presence of Ang II for an additional 4 h. Western blot analysis was performed to observe changes in phospho-Src, phospho-β-catenin, and phospho-VE-cadherin protein expression ( n = 4 for each lane). b Changes in mDia1 protein levels in HUVECs following transfection with RAGE siRNA, as determined by western blotting. Expression was normalized to that of GAPDH ( n = 3 for each lane). c Changes in mDia1 mRNA expression in HUVECs following transfection with RAGE siRNA, as determined by RT-PCR. Expression was normalized to that of the 18S rRNA gene ( n = 4 for each lane). d HUVECs were treated with Ang II and the NF-κB inhibitor (5 μg/ml) alone or in combination for 4 h. Protein levels of AT1R, RAGE, and mDia1 in cell lysates were determined by western blotting. Expression was normalized to that of GAPDH ( n = 4 for each lane). The values are presented as the means ± SEMs. * p

    Journal: Experimental & Molecular Medicine

    Article Title: Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE

    doi: 10.1038/s12276-019-0312-5

    Figure Lengend Snippet: RAGE regulates Ang II-induced endothelial hyperpermeability via mDia1. a HUVECs were transfected with mDia1 siRNA and cultured in the presence of Ang II for an additional 4 h. Western blot analysis was performed to observe changes in phospho-Src, phospho-β-catenin, and phospho-VE-cadherin protein expression ( n = 4 for each lane). b Changes in mDia1 protein levels in HUVECs following transfection with RAGE siRNA, as determined by western blotting. Expression was normalized to that of GAPDH ( n = 3 for each lane). c Changes in mDia1 mRNA expression in HUVECs following transfection with RAGE siRNA, as determined by RT-PCR. Expression was normalized to that of the 18S rRNA gene ( n = 4 for each lane). d HUVECs were treated with Ang II and the NF-κB inhibitor (5 μg/ml) alone or in combination for 4 h. Protein levels of AT1R, RAGE, and mDia1 in cell lysates were determined by western blotting. Expression was normalized to that of GAPDH ( n = 4 for each lane). The values are presented as the means ± SEMs. * p

    Article Snippet: Anti-AT1R antibody (AAR-011) was purchased from Alomone Labs (Hadassah Ein Kerem, Israel).

    Techniques: Transfection, Cell Culture, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    RT-PCR and sequence analysis of porcine P2Y2 , P2Y4 and P2Y6 receptors

    Journal:

    Article Title: Evidence for the Expression of Multiple Uracil Nucleotide-Stimulated P2 Receptors Coupled to Smooth Muscle Contraction in Porcine Isolated Arteries

    doi: 10.1038/sj.bjp.0707120

    Figure Lengend Snippet: RT-PCR and sequence analysis of porcine P2Y2 , P2Y4 and P2Y6 receptors

    Article Snippet: For example, reports describe expression of mRNA for P2Y2 , P2Y4 and P2Y6 receptors in human coronary artery , P2Y2 and P2Y6 receptors in human omental and cerebral arteries ( ) and P2Y2 , P2Y4 and P2Y6 receptors in human placental vasculature ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

    P2Y6 receptor expressed on microglia and dramatically increased within 3 d after tMCAO. A, Western blot and the quantification of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after ischemic stroke. B, Quantitative of RT‐PCR analysis for the expression of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after tMCAO. C, Representative fluorescence images of P2Y6 receptor in the contralateral and ipsilateral hemispheres of mice after tMCAO. Scale bar = 100 μm. D, Z‐stack for representative fluorescence images of (a) P2Y6 receptor (green) and Iba1 (red), (b) P2Y6 receptor (green) and GFAP (red), and (c) P2Y6 receptor (green) and Tuj 1 (red). Scale bar = 10 μm. Low magnification images are also presented respectively. Scale bar = 50 μm. Data were presented as mean ± SEM (n = 3 per group). * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: P2Y6 receptor expressed on microglia and dramatically increased within 3 d after tMCAO. A, Western blot and the quantification of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after ischemic stroke. B, Quantitative of RT‐PCR analysis for the expression of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after tMCAO. C, Representative fluorescence images of P2Y6 receptor in the contralateral and ipsilateral hemispheres of mice after tMCAO. Scale bar = 100 μm. D, Z‐stack for representative fluorescence images of (a) P2Y6 receptor (green) and Iba1 (red), (b) P2Y6 receptor (green) and GFAP (red), and (c) P2Y6 receptor (green) and Tuj 1 (red). Scale bar = 10 μm. Low magnification images are also presented respectively. Scale bar = 50 μm. Data were presented as mean ± SEM (n = 3 per group). * P

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Fluorescence, Mouse Assay

    P2Y6 receptor antagonist MRS2578 inhibit MLCK expression in mice at 3 d after tMCAO and cultured microglia. A, (a) Representative fluorescence images of MLCK in Iba‐1 + cultured primary microglia. Scale bar = 25 μm. (b) Quantitative analysis of MLCK in each Iba1 + microglia. n = 4 per group. B, (a) Western blot for the expression of MLCK in the control, LPS+UDP and LPD+UDP+MRS2578 group. (b) Quantification for the intensity ratios of MLCK/β‐actin in the control, LPS+UDP and LPD+UDP+MRS2578 group. C, (a) Western blot for the expression of MLCK in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification for the intensity ratios of MLCK/β‐actin in the sham, saline, and MRS2578 group. Data were presented as mean ± SEM. n = 3‐4 per group. * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: P2Y6 receptor antagonist MRS2578 inhibit MLCK expression in mice at 3 d after tMCAO and cultured microglia. A, (a) Representative fluorescence images of MLCK in Iba‐1 + cultured primary microglia. Scale bar = 25 μm. (b) Quantitative analysis of MLCK in each Iba1 + microglia. n = 4 per group. B, (a) Western blot for the expression of MLCK in the control, LPS+UDP and LPD+UDP+MRS2578 group. (b) Quantification for the intensity ratios of MLCK/β‐actin in the control, LPS+UDP and LPD+UDP+MRS2578 group. C, (a) Western blot for the expression of MLCK in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification for the intensity ratios of MLCK/β‐actin in the sham, saline, and MRS2578 group. Data were presented as mean ± SEM. n = 3‐4 per group. * P

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Expressing, Mouse Assay, Cell Culture, Fluorescence, Western Blot

    Inhibition of P2Y6 receptor activity reduced the phagocytic function of microglia after tMCAO. A, Representative fluorescence images of Iba1 + microglia (red) and TUNEL + apoptotic cells (green) in the peri‐infarct region of brain slice in the sham (a), saline (b), and MRS2578 (c) group at 3 d after tMCAO. Scale bar = 50 μm. White arrows indicated Iba1 + cells engulfed TUNEL + cells in the saline and MRS2578 group. B, Semiquantitative analysis of Iba1 + microglial phagocytosis for TUNEL + cells. n = 4. C, Representative fluorescence images of Iba1 + microglia (green), lysosome marker LAMP‐2 (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. 3D scanning with depth indicated Iba1 + cells phagocytosed TUNEL + cells and transferred them into lysosomes. Scale bar = 5 μm. D, Representative fluorescence image of Iba1 + microglia (green), NeuN + neurons (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. Scale bar = 5 μm. 3D scanning with depth indicated Iba1 + cells engulfed TUNEL + neurons

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: Inhibition of P2Y6 receptor activity reduced the phagocytic function of microglia after tMCAO. A, Representative fluorescence images of Iba1 + microglia (red) and TUNEL + apoptotic cells (green) in the peri‐infarct region of brain slice in the sham (a), saline (b), and MRS2578 (c) group at 3 d after tMCAO. Scale bar = 50 μm. White arrows indicated Iba1 + cells engulfed TUNEL + cells in the saline and MRS2578 group. B, Semiquantitative analysis of Iba1 + microglial phagocytosis for TUNEL + cells. n = 4. C, Representative fluorescence images of Iba1 + microglia (green), lysosome marker LAMP‐2 (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. 3D scanning with depth indicated Iba1 + cells phagocytosed TUNEL + cells and transferred them into lysosomes. Scale bar = 5 μm. D, Representative fluorescence image of Iba1 + microglia (green), NeuN + neurons (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. Scale bar = 5 μm. 3D scanning with depth indicated Iba1 + cells engulfed TUNEL + neurons

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Inhibition, Activity Assay, Fluorescence, TUNEL Assay, Slice Preparation, Marker

    P2Y6 receptor‐specific inhibitor MRS2578 blocked the phagocytosis of primary microglia under LPS UDP stimulation. A, Representative images of fluorescent latex beads (yellow) phagocytized by primary microglia under 12 h LPS (200 ng/mL) UDP (100 μmol/L) stimulation in the control, LPS+UDP, 1 μmol/L MRS, 2 μmol/L MRS, and 5 μmol/L MRS group. (a) Control group, only cultured by basic culture medium of microglia, (b) MRS2578‐free group, 200 ng/mL LPS, and 100 μmol/L UDP in basic culture medium of microglia, (c) 1 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 1 μmol/L MRS2578 in basic culture medium of microglia, (d) 2 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 2 μmol/L MRS2578 in basic culture medium of microglia, (e) 5 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 5 μmol/L MRS2578 in basic culture medium of microglia. Scale bar = 10 μm. B, Quantification for the number of beads per microglia under LPS (200 ng/mL) UDP (100 μmol/L) stimulation (n = 3 per group). C, CCK‐8 assay in primary microglia without or with different concentrations MRS2578 under LPS+UDP stimulation for 12 h. D, (a) Representative flow cytometry analysis of microglia phagocytosis. Phagocytosis of fluorescein isothiocyanate (FITC)‐labeled latex beads by CD206 + and CD206 ‐ microglia. (Green, untreated control; Red, LPS+UDP treatment; Blue: LPS+UDP+1 μmol/L MRS2578 treatment); (b) Percentage of FITC + cells in CX 3 CR 1 + cells for total, CD206+, and CD206‐ microglia. E, (a) Representative diagrams of CD206 + /CX 3 CR 1 + cells in the control, LPS+UDP, LPS+UDP+MRS2578 group, (b) Percentage of CD206 + /CX 3 CR 1 + in the control, LPS+UDP, LPS+UDP+MRS2578 group. Data were presented as mean ± SEM. n = 3 per group. * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: P2Y6 receptor‐specific inhibitor MRS2578 blocked the phagocytosis of primary microglia under LPS UDP stimulation. A, Representative images of fluorescent latex beads (yellow) phagocytized by primary microglia under 12 h LPS (200 ng/mL) UDP (100 μmol/L) stimulation in the control, LPS+UDP, 1 μmol/L MRS, 2 μmol/L MRS, and 5 μmol/L MRS group. (a) Control group, only cultured by basic culture medium of microglia, (b) MRS2578‐free group, 200 ng/mL LPS, and 100 μmol/L UDP in basic culture medium of microglia, (c) 1 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 1 μmol/L MRS2578 in basic culture medium of microglia, (d) 2 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 2 μmol/L MRS2578 in basic culture medium of microglia, (e) 5 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 5 μmol/L MRS2578 in basic culture medium of microglia. Scale bar = 10 μm. B, Quantification for the number of beads per microglia under LPS (200 ng/mL) UDP (100 μmol/L) stimulation (n = 3 per group). C, CCK‐8 assay in primary microglia without or with different concentrations MRS2578 under LPS+UDP stimulation for 12 h. D, (a) Representative flow cytometry analysis of microglia phagocytosis. Phagocytosis of fluorescein isothiocyanate (FITC)‐labeled latex beads by CD206 + and CD206 ‐ microglia. (Green, untreated control; Red, LPS+UDP treatment; Blue: LPS+UDP+1 μmol/L MRS2578 treatment); (b) Percentage of FITC + cells in CX 3 CR 1 + cells for total, CD206+, and CD206‐ microglia. E, (a) Representative diagrams of CD206 + /CX 3 CR 1 + cells in the control, LPS+UDP, LPS+UDP+MRS2578 group, (b) Percentage of CD206 + /CX 3 CR 1 + in the control, LPS+UDP, LPS+UDP+MRS2578 group. Data were presented as mean ± SEM. n = 3 per group. * P

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Cell Culture, CCK-8 Assay, Flow Cytometry, Labeling

    Inhibition of P2Y6 receptor activity exacerbated neurological function deficit and brain injury after tMCAO in mice. A, (a) Representative images of cresyl violet staining for brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification of brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (c) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. B, (d) Representative images of cresyl violet staining for brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (e) Quantification of brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (f) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. C. mNSS in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. mNSS = modified neurological severity scores. D, Grid walking test in the saline and MRS2578 group at 3, 7, and 14 d after tMCAO. E, Body weight in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. F, Survival curves of the saline and MRS2578 group within 3 d after tMCAO. Data were presented as mean ± SEM. n = 6‐11 per group. * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: Inhibition of P2Y6 receptor activity exacerbated neurological function deficit and brain injury after tMCAO in mice. A, (a) Representative images of cresyl violet staining for brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification of brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (c) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. B, (d) Representative images of cresyl violet staining for brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (e) Quantification of brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (f) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. C. mNSS in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. mNSS = modified neurological severity scores. D, Grid walking test in the saline and MRS2578 group at 3, 7, and 14 d after tMCAO. E, Body weight in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. F, Survival curves of the saline and MRS2578 group within 3 d after tMCAO. Data were presented as mean ± SEM. n = 6‐11 per group. * P

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Inhibition, Activity Assay, Mouse Assay, Staining, Modification

    Inhibiting the P2Y6 receptor activity had no effect on the expression of inflammatory cytokines and neutrophil infiltration. A, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10 (d), TNF‐α (e), and TGF‐β (f) at 3 d after tMCAO in the sham, saline, and MRS2578 group. The data in sham group were normalized to 1. B, Western blot for the expression of MPO in the brain of sham, saline, and MRS2578 groups at 3 d after tMCAO (g). Quantification of the intensity ratios of MPO/ β‐actin (h) in the sham, saline, and MRS2578 group. The data in the saline group were normalized to 1. C, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10(d), TNF‐α (e), and TGF‐β (f) after 1 h OGD and 11 h reoxygenation in the control, OGD and OGD+MRS2578 group. The data in the control group were normalized to 1. Data were presented as mean ± SEM. n = 3‐4 per group. * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

    doi: 10.1111/cns.13296

    Figure Lengend Snippet: Inhibiting the P2Y6 receptor activity had no effect on the expression of inflammatory cytokines and neutrophil infiltration. A, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10 (d), TNF‐α (e), and TGF‐β (f) at 3 d after tMCAO in the sham, saline, and MRS2578 group. The data in sham group were normalized to 1. B, Western blot for the expression of MPO in the brain of sham, saline, and MRS2578 groups at 3 d after tMCAO (g). Quantification of the intensity ratios of MPO/ β‐actin (h) in the sham, saline, and MRS2578 group. The data in the saline group were normalized to 1. C, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10(d), TNF‐α (e), and TGF‐β (f) after 1 h OGD and 11 h reoxygenation in the control, OGD and OGD+MRS2578 group. The data in the control group were normalized to 1. Data were presented as mean ± SEM. n = 3‐4 per group. * P

    Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

    Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot