anti gpr91  (Alomone Labs)

Journal: bioRxiv

Article Title: Airway succinate chemosensing induces CFTR-dependent anion secretion and mucus clearance which is impaired in cystic fibrosis

doi: 10.1101/2024.03.26.586799

Figure Lengend Snippet: ( A ) Summary of the changes in short-circuit current (I SC ) in mouse trachea in response to the indicated di-carboxylates, representative traces of I SC in mouse tracheas stimulated with increasing succinate concentrations and a summary of succinate-induced I SC in mouse tracheas. ( B ) Representative trace of succinate-, CCh- and UTP-induced anion secretion in wild type, Sucnr1 -/- tracheas and wild type tracheas incubated with TPPO and 4-DAMP. ( C ) Summary of succinate-induced, CCh-induced and UTP-induced anion secretion in mouse tracheas treated with TPPO, 4-DAMP or pirenzipine; values are Mean ± S.E.M.; n=3-5 per group.* indicates p<0.05, ANOVA on Ranks.

Article Snippet: Detection of SUCNR1 was performed using the anti-GPR91 (SUCNR1; Alomone #ASR-090) and TRPM5 with anti-TRPM5 (Alomone, #ACC-054), both tested with and without antigen retrieval using pH 6.0 or pH 8.0 buffers and overnight incubation at RT or 4 C°.

Techniques: Incubation

Journal: bioRxiv

Article Title: Airway succinate chemosensing induces CFTR-dependent anion secretion and mucus clearance which is impaired in cystic fibrosis

doi: 10.1101/2024.03.26.586799

Figure Lengend Snippet: ( A ) Mucus transport speed before and after succinate in wild type, Sucnr1 -/- and wild type tracheas treated with 30 nM 4-DAMP. Values are Mean ± S.E.M.; n=4 for each group; Rank-sum test. ( B ) Representative traces for succinate induced I SC in control, after 30 µM ANI9 of 30 µM S0859 and summary of succinate-induced ΔI SC , values are Mean ± S.E.M.; n=4 for each group; ANOVA on Ranks. ( C ) Succinate-induced changes in mucus transport speed was determined in wild type mice after 30 µM ANI9 or 30 µM S0859 or in Slc4a4 +/+ and Slc4a4 -/- tracheas. Values are Mean ± S.E.M.; n=4 for each group; Rank-sum test. ( D ) Representative traces of bronchoconstriction in wild type (blue line) or Sucnr1 -/- (red line) tracheal rings after 1mM succinate and 10 µM carbachol (CCh) addition followed by representative images of tracheal rings; bars = 500 µm. Numbers indicate the moment where corresponding images where taken. Summary of bronchoconstriction experiments, blue dots for wild type and red dots for Sucnr1 -/- . Bars are values are Mean ± S.E.M.; n=5 for each group; Rank-sum test.

Article Snippet: Detection of SUCNR1 was performed using the anti-GPR91 (SUCNR1; Alomone #ASR-090) and TRPM5 with anti-TRPM5 (Alomone, #ACC-054), both tested with and without antigen retrieval using pH 6.0 or pH 8.0 buffers and overnight incubation at RT or 4 C°.

Techniques: Control

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: The expression of SUCNR1 in cells from Ectopic milieu. A SUCNR1 expression in female tissues from healthy human tissues (data are available from https://www.proteinatlas.org/). B-D The level of SUCNR1 + macrophages and peritoneal mesothelial cells (PMC) in peritoneal fluid from EMs patients was elevated and correlated with EMs stage. E The staining macroscopic observation of peritoneal mesothelial cells (PMC) in peritoneal fluid or intraperitoneal lavage (modified papanicolaou staining, 40X). F-G FCM-based MFI assay and qT-PCR analysis designed to measure SUCNR1 expressions, and result showed primary ectopic stromal cell (hESC.D) expressed higher SUCNR1, compared with that of primary normal stromal cell (hESC.N) and human stromal cell line (hESC.N). H-I FCM and qT-PCR assays showed the SUCNR1 expression of primary normal ESC stimulated with succiante with different concentrations. The optimal concentration of succinate is 2.5 mM. Significance was assessed either with t test or one-way ANOVA followed by Tukey’s or Holm-Sidak’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Peritoneal cell pellets collected from the peritoneal fluid were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325,621), anti-human CD45 APC-Cy7 (BioLegend, 368,515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F).

Techniques: Expressing, Staining, Modification, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: Succinate Induce the Enrichment of SUCNR1 + Macrophages and Ectopic Lesion Formation in vivo . A The wight change of mouse EMs model during the 2 weeks. Ctrl: PBS treatment; M: Model group; M + S: Model with succinate treatment group. The data are expressed as the mean ± SEM. (One-way ANOVA) * P < 0.05, ** P < 0.01 and *** P < 0.001. B The macroscopic observation of the morphology of endometriosis-like lesions from endometriosis mouse models. M: Model group, M + S: Model with succinate treatment group. C-D The number and wight of EMs lesions was measured after the treatment of succinate (100 mg/kg) or PBS. M: Model group, M + S: Model with succinate treatment group. ( Student’s t-test ) * P < 0.05, ** P < 0.01 and *** P < 0.001. E-I The levels of CD80, CD163 and SUCNR1 on peritoneal CD11b(+)F4/80(+) macrophages from mouse models were analyzed by using flow cytometry. MFI of SUCNR1 on M1 and M2 macrophages obtained were shown respectively. Ctrl: marked in red; M: marked in blue; M + S: marked in orange. ( One-way ANOVA ) * P < 0.05, ** P < 0.01 and *** P < 0.001. J MMP9 and ICAM-1 expression in endometriosis-like lesion from model mice (M) and succinate exposing mice (M + S) by immunohistochemistry. M: Model group; M + S: Model with succinate treatment group. Original magnification: × 200

Article Snippet: Peritoneal cell pellets collected from the peritoneal fluid were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325,621), anti-human CD45 APC-Cy7 (BioLegend, 368,515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F).

Techniques: In Vivo, Flow Cytometry, Expressing, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: Schematic roles of Succinate from macrophage and peritoneal mesothelial cells in the progression of endometriosis by inducing inflammation and promoting ectopic growth. In the microenvironment of ectopic foci, exposure to inflammatory cytokines or contaction with ESCs obviously triggered succinate secretion from M1 polarized macrophages and PMCs, which are the main producers of succinate. Interestingly, compared with the normal endometrium, ESCs in ectopic tissues express high levels of SUCNR1. In PMCs, succinate promotes the auto-secretion of succinate and the expression of SUCNR1 in an autocrine amplification manner. In addition to promoting succinate accumulation, interactions between macrophages, mesothelial cells and ESCs in the ectopic milieu amplify the regulatory effect on cellular function via SUCNR1 signaling. Succinate promoted the survival, adhesion, invasion, and deep infiltration of ESCs via SUCNR1 signaling, leading to the formation of ectopic lesions in endometriosis. In conclusion, succinate in the ectopic milieu synergizes with polarized macrophages to exacerbate inflammation and facilitate endometriosis progress via succinate-SUCNR1-dependent mechanisms

Article Snippet: Peritoneal cell pellets collected from the peritoneal fluid were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325,621), anti-human CD45 APC-Cy7 (BioLegend, 368,515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F).

Techniques: Expressing, Amplification, Cell Function Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: The expression of SUCNR1 in cells from Ectopic milieu. A SUCNR1 expression in female tissues from healthy human tissues (data are available from https://www.proteinatlas.org/). B-D The level of SUCNR1 + macrophages and peritoneal mesothelial cells (PMC) in peritoneal fluid from EMs patients was elevated and correlated with EMs stage. E The staining macroscopic observation of peritoneal mesothelial cells (PMC) in peritoneal fluid or intraperitoneal lavage (modified papanicolaou staining, 40X). F-G FCM-based MFI assay and qT-PCR analysis designed to measure SUCNR1 expressions, and result showed primary ectopic stromal cell (hESC.D) expressed higher SUCNR1, compared with that of primary normal stromal cell (hESC.N) and human stromal cell line (hESC.N). H-I FCM and qT-PCR assays showed the SUCNR1 expression of primary normal ESC stimulated with succiante with different concentrations. The optimal concentration of succinate is 2.5 mM. Significance was assessed either with t test or one-way ANOVA followed by Tukey’s or Holm-Sidak’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305,419); anti-human/mouse MCT1 PE (R&D, FAB8275P); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103,129); anti-human/mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD80 PE (BioLegend, 104,707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105,024); anti-mouse CD163 BV421 (BioLegend, 155,309); anti-mouse CD206 BV605 (BioLegend, 141,721), anti-mouse CD11b PCy 7(BioLegend, 101,215); anti-mouse F4/80 APC (BioLegend, 123,116).

Techniques: Expressing, Staining, Modification, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: Succinate Induce the Enrichment of SUCNR1 + Macrophages and Ectopic Lesion Formation in vivo . A The wight change of mouse EMs model during the 2 weeks. Ctrl: PBS treatment; M: Model group; M + S: Model with succinate treatment group. The data are expressed as the mean ± SEM. (One-way ANOVA) * P < 0.05, ** P < 0.01 and *** P < 0.001. B The macroscopic observation of the morphology of endometriosis-like lesions from endometriosis mouse models. M: Model group, M + S: Model with succinate treatment group. C-D The number and wight of EMs lesions was measured after the treatment of succinate (100 mg/kg) or PBS. M: Model group, M + S: Model with succinate treatment group. ( Student’s t-test ) * P < 0.05, ** P < 0.01 and *** P < 0.001. E-I The levels of CD80, CD163 and SUCNR1 on peritoneal CD11b(+)F4/80(+) macrophages from mouse models were analyzed by using flow cytometry. MFI of SUCNR1 on M1 and M2 macrophages obtained were shown respectively. Ctrl: marked in red; M: marked in blue; M + S: marked in orange. ( One-way ANOVA ) * P < 0.05, ** P < 0.01 and *** P < 0.001. J MMP9 and ICAM-1 expression in endometriosis-like lesion from model mice (M) and succinate exposing mice (M + S) by immunohistochemistry. M: Model group; M + S: Model with succinate treatment group. Original magnification: × 200

Article Snippet: The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305,419); anti-human/mouse MCT1 PE (R&D, FAB8275P); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103,129); anti-human/mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD80 PE (BioLegend, 104,707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105,024); anti-mouse CD163 BV421 (BioLegend, 155,309); anti-mouse CD206 BV605 (BioLegend, 141,721), anti-mouse CD11b PCy 7(BioLegend, 101,215); anti-mouse F4/80 APC (BioLegend, 123,116).

Techniques: In Vivo, Flow Cytometry, Expressing, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

doi: 10.1186/s12964-023-01415-7

Figure Lengend Snippet: Schematic roles of Succinate from macrophage and peritoneal mesothelial cells in the progression of endometriosis by inducing inflammation and promoting ectopic growth. In the microenvironment of ectopic foci, exposure to inflammatory cytokines or contaction with ESCs obviously triggered succinate secretion from M1 polarized macrophages and PMCs, which are the main producers of succinate. Interestingly, compared with the normal endometrium, ESCs in ectopic tissues express high levels of SUCNR1. In PMCs, succinate promotes the auto-secretion of succinate and the expression of SUCNR1 in an autocrine amplification manner. In addition to promoting succinate accumulation, interactions between macrophages, mesothelial cells and ESCs in the ectopic milieu amplify the regulatory effect on cellular function via SUCNR1 signaling. Succinate promoted the survival, adhesion, invasion, and deep infiltration of ESCs via SUCNR1 signaling, leading to the formation of ectopic lesions in endometriosis. In conclusion, succinate in the ectopic milieu synergizes with polarized macrophages to exacerbate inflammation and facilitate endometriosis progress via succinate-SUCNR1-dependent mechanisms

Article Snippet: The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305,419); anti-human/mouse MCT1 PE (R&D, FAB8275P); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103,129); anti-human/mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD80 PE (BioLegend, 104,707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105,024); anti-mouse CD163 BV421 (BioLegend, 155,309); anti-mouse CD206 BV605 (BioLegend, 141,721), anti-mouse CD11b PCy 7(BioLegend, 101,215); anti-mouse F4/80 APC (BioLegend, 123,116).

Techniques: Expressing, Amplification, Cell Function Assay