Journal: Gut Microbes

Article Title: Cerebrovascular damage caused by the gut microbe/host co-metabolite p -cresol sulfate is prevented by blockade of the EGF receptor

doi: 10.1080/19490976.2024.2431651

Figure Lengend Snippet: pCS enhances endothelial permeability via mobilization of active MMP2 and MMP9. (a) Treatment of hCMEC/D3 cells with pCS (10 µm, 24 h) increases expression of MMP2 and MMP9; data are mean ± s.e.m., n = 7 independent experiments. (b) stimulation of MMP2 or MMP9 expression in hCMEC/D3 cells by pCS (10 µm, 24 h) is prevented by either erlotinib (2.5 µm, 10 min pre-treatment) or C188-9 (5 µm, 30 min pre-treatment); data are mean ± s.e.m., n = 6 independent experiments. (c) active MMP2 and MMP9 are secreted into the culture medium of hCMEC/D3 cells in response to pCS (10 µm, 24 h) stimulation; data are mean ± s.e.m., n = 6 independent experiments; a typical gelatin zymography is shown, highlighting both active MMP2 and MMP9 and artifactually revealed pro-MMP2 and pro-MMP9 activity. (d) active MMP2 and MMP9 release from hCMEC/D3 cells into culture medium in response to pCS (10 µm, 24 h) stimulation is prevented by erlotinib (2.5 µm, 10 min pre-treatment); data are mean ± s.e.m., n = 11-12 independent replicates. e) pre-treatment of hCMEC/D3 cell monolayers with the MMP2 inhibitor ARP 100 (12 nM, 15 min) prevents the increase in paracellular permeability to a 70 kDa fitc-dextran conjugate induced by pCS stimulation (10 µm, 24 (h); data are mean ± s.e.m., n = 6 independent experiments. (f) pre-treatment of hCMEC/D3 cell monolayers with ARP 100 (12 nM, 15 min) prevents the reduction in TEER induced by pCS stimulation (10 µm, 24 h); data are mean ± s.e.m., n = 6 independent experiments. (g) confocal microscopic analysis of expression of the tight junction component zona occludens-1 (ZO-1; white) in hCMEC/D3 cells following treatment with either pCS (10 µm, 24 h), ARP 100 (12 nM, 24 h) or both (ARP 100 12 nM, 15 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale = 15 µm. Images are representative of at least three independent experiments. (h) pre-treatment of hCMEC/D3 cell monolayers with the MMP9 inhibitor JNJ0966 (440 nM, 15 min) prevents the increase in paracellular permeability to a 70 kDa fitc-dextran conjugate induced by pCS stimulation (10 µm, 24 h); data are mean ± s.e.m., n = 6 independent experiments. (i) pre-treatment of hCMEC/D3 cell monolayers with JNJ0966 (440 nM, 15 min) prevents the reduction in TEER induced by pCS stimulation (10 µm, 24 h); data are mean ± s.e.m., n = 6 independent experiments. (j) confocal microscopic analysis of expression of the tight junction component zona occludens-1 (ZO-1; white) in hCMEC/D3 cells following treatment with either pCS (10 µm, 24 h), JNJ0966 (440 nM, 24 h) or both (JNJ0966 440 nM, 15 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale = 15 µm. Images are representative of at least three independent experiments.

Article Snippet: Primary antibodies used were MMP2: rabbit anti-human, 1:1000 Insight Biotechnology Ltd., UK, MMP9: mouse anti-human, 1:50, Santa Cruz Biotechnology Inc, USA, phosphotyrosine-21 ANXA1: rabbit anti-human, 1:1000, ZO-1: rabbit anti-human 1:100, Thermofisher Scientific, UK.

Techniques: Permeability, Expressing, Zymography, Activity Assay

Journal: Gut Microbes

Article Title: Cerebrovascular damage caused by the gut microbe/host co-metabolite p -cresol sulfate is prevented by blockade of the EGF receptor

doi: 10.1080/19490976.2024.2431651

Figure Lengend Snippet: Treatment with pCS induces BBB permeability in vivo throughEGFR-dependent mobilization of MMP9. a, b) Confocal microscopic analysis of expression of (a) MMP-2 or (b) MMP-9 (magenta) in the brains of male C57Bl/6 exposed to pCS by s.C. implantation with osmotic mini-pumps releasing at 7.5 µg/h for 28 days, blood vessels are defined by tomato lectin binding (cyan), nuclei are counterstained with DAPI (white), scale = 10 µm, images are representative of 7-10 animals. c, d) confocal microscopic analysis of (c) MMP2 and (d) MMP9 expression (magenta) within the cerebral microvasculature of male C57Bl/6 mice exposed to pCS (10 mg/kg, i.P. 2 h) with or without erlotinib pre-treatment (50 mg/kg, i.P. 1 h pre-treatment); blood vessels are defined by tomato lectin binding (cyan), nuclei are counterstained with DAPI (white), scale bar = 5 µm, images are representative of 7-10 animals. (e) typical expression of ZO-1 (white) within the cerebral microvasculature of male C57Bl/6 mice exposed to pCS (10 mg/kg, i.P. 2 h) with or without erlotinib pre-treatment (50 mg/kg, i.P. 1 h pre-treatment); nuclei are counterstained with DAPI (blue), scale bar = 10 µm. (f) Increased extravasation of Evans blue tracer into the parenchyma of male C57Bl/6 mice following i.p. injection of pCS (10 mg/kg, 2 h exposure) is prevented by erlotinib administration (50 mg/kg i.p., 1 h pre-treatment); data are mean ± s.e.m., n = 5–6 animals. (g) Increased extravasation of Evans blue tracer into the parenchyma of male C57Bl/6 mice following i.p. injection of pCS (10 mg/kg, 2 h exposure) is prevented by administration of the dual-specific MMP2/MMP9 inhibitor SB-3CT (10 mg/kg i.p., 1 h pre-treatment); data are mean ± s.e.m., n = 5–6 animals.

Article Snippet: Primary antibodies used were MMP2: rabbit anti-human, 1:1000 Insight Biotechnology Ltd., UK, MMP9: mouse anti-human, 1:50, Santa Cruz Biotechnology Inc, USA, phosphotyrosine-21 ANXA1: rabbit anti-human, 1:1000, ZO-1: rabbit anti-human 1:100, Thermofisher Scientific, UK.

Techniques: Permeability, In Vivo, Expressing, Binding Assay, Injection

Journal: Gut Microbes

Article Title: Cerebrovascular damage caused by the gut microbe/host co-metabolite p -cresol sulfate is prevented by blockade of the EGF receptor

doi: 10.1080/19490976.2024.2431651

Figure Lengend Snippet: Sera from human hemodialysis patients significantly enhances endothelial permeability, an effect prevented by erlotinib pre-treatment. (a) Treatment of hCMEC/D3 monolayers with medium containing de-complemented sera from patients undergoing hemodialysis (20%, 24 h) significantly enhances permeability to a 70 kDa fitc-dextran tracer, an effect not seen with equivalent sera from healthy donors, and prevented by inclusion of erlotinib (2.5 µm, 10 min); data are mean ± s.e.m., n = 10-11 donors. (b) TEER of hCMEC/D3 monolayers is not affected by treatment with media containing sera (20%, 24 h) from either hemodialysis patients or healthy donors, nor by erlotinib inclusion (2.5 µm, 10 min pre-treatment); data are mean ± s.e.m., n = 10-11 donors. (c) treatment of hCMEC/D3 cells with medium containing de-complemented sera (20%, 24 h) from patients undergoing hemodialysis, but not with that from healthy donors, increases expression of MMP2; data are mean ± s.e.m., n = 10-11 donors. (d) treatment of hCMEC/D3 cells with medium containing de-complemented sera (20%, 24 h) from patients undergoing hemodialysis, but not with that from healthy donors, increases expression of MMP9; data are mean ± s.e.m., n = 10-11 donors. (e) correlation between concentrations of pCS in human sera samples (healthy donors in black, hemodialysis patients in red) with 70 kDa fitc-dextran permeability data from (A) above; n = 10-11 donors. (f) correlation between concentrations of xanthurenic acid in human sera samples (healthy donors in black, hemodialysis patients in red) with 70 kDa fitc-dextran permeability data from (A) above; n = 10-11 donors.

Article Snippet: Primary antibodies used were MMP2: rabbit anti-human, 1:1000 Insight Biotechnology Ltd., UK, MMP9: mouse anti-human, 1:50, Santa Cruz Biotechnology Inc, USA, phosphotyrosine-21 ANXA1: rabbit anti-human, 1:1000, ZO-1: rabbit anti-human 1:100, Thermofisher Scientific, UK.

Techniques: Permeability, Expressing

Journal: Journal of Cardiovascular Development and Disease

Article Title: New Insights on the Formation of the Mitral Valve Chordae Tendineae in Fetal Life

doi: 10.3390/jcdd11110367

Figure Lengend Snippet: Antibody specific differences in immunohistochemistry protocol.

Article Snippet: Primary Antibody and Dilution , CD31 rabbit polyclonal (Abcam, Cambridge, UK, ab28364, 1:50) , MMP-1 rabbit monoclonal (Abcam ab52631, 1:40) , MMP-2 rabbit polyclonal (Abcam ab97779, 1:200) , Ki-67 rabbit monoclonal (Abcam ab16667, 1:200).

Techniques: Immunohistochemistry, Staining, Incubation, Polymer

Journal: Discover Oncology

Article Title: POLQ knockdown inhibits proliferation, migration, and invasion by inducing cell cycle arrest in colorectal cancer

doi: 10.1007/s12672-024-01496-x

Figure Lengend Snippet: POLQ silencing affects the cell cycle signaling pathway in CRC ( A , B , C ) WB was used to detect the expression of CDK6, CDK4, cyclin D1, MCM2, MCM7, MMP2, and MMP9 in negative control (NC) cells and POLQ-knockdown HCT116 and SW480 cells. D CCK8 assay was used to assess the proliferation of HCT116 cells treated with various concentrations of agonist. E CCK8 assay was used to detect the effect of agonist treatment and POLQ knockdown on the proliferation of HCT116 cells. F , G Colony formation assay was used to analyze the proliferation ability of HCT116 cells after agonist treatment and POLQ knockdown. H The transcriptional level of POLQ in HCT116 cells treated with agonist and siRNAs was measured by qRT‒PCR. I The expression levels of POLQ, CDK6, CDK4, cyclin D1, MCM2 and MCM7 were assessed by WB after treatment with agonist and siRNAs in HCT116 cells. *P < 0.01, ***P < 0.001, ****P < 0.0001. (Control group: none; DMSO group: DMSO only; SI group: siRNAs only; AG group: agonist + DMSO; SI + AG group: siRNAs + agonist + DMSO)

Article Snippet: Cat no. 220965, Sangon Biotech), rabbit anti-MMP2 (1:1000.

Techniques: Expressing, Negative Control, Knockdown, CCK-8 Assay, Colony Assay, Control