af488 conjugated goat anti rabbit  (Thermo Fisher)

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: Exposure to pCS increases the permeability of human cerebromicrovascular endothelial cells in vitro and the murine BBB in vivo . (a) Incubation of hCMEC/D3 cell monolayers with pCS (10 µm, 100 µm, 1 mm; 24 h) dose-dependently increased paracellular permeability to a 70 kDa fitc-dextran conjugate; data are mean ± s.e.m., n = 9 independent experiments. (b) incubation of hCMEC/D3 cell monolayers with pCS (10 µm, 100 µm, 1 mm; 24 h) dose-dependently reduced TEER; data are mean ± s.e.m., n = 9 independent experiments. (c, d) confocal microscopic analysis of expression of C) AF488-phalloidin labeled actin filaments (white) or d) the tight junction component zona occludens-1 (ZO-1; white) in hCMEC/D3 cells following treatment for 24 h with 10 µm pCS, nuclei are counterstained with DAPI (blue), scale = 15 µm. Images are representative of at least three independent experiments. (e) treatment of male C57Bl/6 mice by i.P. injection of pCS (10 mg/kg) caused a time-dependent increase in extravasation of Evans blue tracer into the CNS parenchyma, reaching statistical significance at both 2 h and 6 h post administration; data are mean ± s.e.m., n = 5-6 animals. (f) exposure of male C57Bl/6 by s.C. implantation with osmotic mini-pumps releasing pCS at 7.5 µg/h for 28 days significantly enhanced extravasation of sodium fluorescein into the brain parenchyma; data are mean ± s.e.m., n = 7-10 animals. (g, h) confocal microscopic analysis of expression of G) claudin-5 or H) zonula occludens-1 (ZO-1) in the brains of male C57Bl/6 exposed by s.C. implantation with osmotic mini-pumps releasing pCS at 7.5 µg/h for 28 days, nuclei are counterstained with DAPI (blue), scale = 10 µm. Images are representative of 7-10 animals; (i) volcano plot showing the 380 significantly differentially expressed genes among the 16,988 mouse genes examined in this study ( n = 5 animals per group (treated, negative control)). Black data points: no significant change in expression; red dots, significantly ( p < 0.05, Benjamini-Hochberg) upregulated expression in the pCS group compared with the negative control; blue dots, significantly ( p < 0.05, Benjamini-Hochberg) downregulated expression in the pCS group compared with the negative control; n = 5 per group. (j) biological processes (above) and molecular functions (below) of genes found to be significantly downregulated ( n = 289) upon exposure of mice to pCS, based on enrichr p value ranking from gene ontology analysis. (k) topological analysis of the KEGG network resulting from mapping the 380 significantly ( p < 0.05, Benjamini-Hochberg) differentially expressed genes onto the 17 KEGG Mus musculus metabolic pathways listed in . Blue solid circles, genes whose expression is significantly downregulated in the pCS group compared with the control; red solid circles, genes whose expression is significantly upregulated in the pCS group compared with the control. Thick outline colors around the solid circles correspond to the molecular functions listed in the legend. Network summary statistics are available in table S3.

Article Snippet: Secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse 1:500, both Stratech Scientific, UK, AF488-conjugated goat anti-rabbit or anti-mouse 1:500, both Thermofisher Scientific, UK.

Techniques: Permeability, In Vitro, In Vivo, Incubation, Expressing, Labeling, Injection, Negative Control, Control

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 regulates cerebromicrovascular cell permeability in vitro through activation of the EGFR. (a) Stimulation of hCMEC/D3 cells with pCS (24 h, 10 µm) has no effect on cell surface expression of EGFR; data are mean ± s.e.m., n = 4 independent experiments; representative flow cytometry histograms are shown. (b) treatment of hCMEC/D3 cells with pCS (10 µm) causes a time-dependent increase in EGFR Tyr1068 phosphorylation, maintained for at least 30 min; data are mean ± s.e.m., n = 9 independent experiments; representative flow cytometry histograms are shown. (c) pre-treatment of hCMEC/D3 cells with erlotinib (2.5 µm, 10 min) prevents EGFR Tyr1068 phosphorylation in response to pCS treatment (10 µm, 30 min), data are mean ± s.e.m., n = 6 independent experiments; representative flow cytometry histograms are shown. (d) pre-treatment of hCMEC/D3 cell monolayers with erlotinib (2.5 µm, 10 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 = 9 independent experiments. (e) pre-treatment of hCMEC/D3 cell monolayers with erlotinib (2.5 µm, 10 min) prevents the reduction in TEER induced by pCS stimulation (10 µm, 24 h); data are mean ± s.e.m., n = 9 independent experiments. (f) 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), erlotinib (2.5 µm, 24 h) or both (erlotinib 2.5 µm, 10 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale bar = 15 µm. Images are representative of at least three independent experiments. (g) confocal microscopic analysis of expression of AF488-conjugated phalloidin labeled actin filaments (white) in hCMEC/D3 cells following treatment with either pCS (10 µm, 24 h), erlotinib (2.5 µm, 24 h) or both (erlotinib 2.5 µm, 10 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale bar = 15 µm. Images are representative of at least three independent experiments. (h) paracellular permeability to a 70 kDa fitc-dextran conjugate of hCMEC/D3 cell monolayers bearing siRNA sequences targeting the EGFR (siRNA 5, siRNA 11, siRNA 12), a non-targeting negative control siRNA sequence, or that had been mock-transfected, with or without stimulation with pCS (10 µm, 24 h); data are mean ± s.e.m., n = 4 independent experiments. (i) TEER of hCMEC/D3 cell monolayers bearing siRNA sequences targeting the EGFR (siRNA 5, siRNA 11, siRNA 12), a non-targeting negative control siRNA sequence, or that had been mock-transfected, with or without stimulation with pCS (10 µm, 24 h); data are mean ± s.e.m., n = 3 independent experiments.

Article Snippet: Secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse 1:500, both Stratech Scientific, UK, AF488-conjugated goat anti-rabbit or anti-mouse 1:500, both Thermofisher Scientific, UK.

Techniques: Permeability, In Vitro, Activation Assay, Expressing, Flow Cytometry, Labeling, Negative Control, Sequencing, Transfection

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: Exposure to pCS increases the permeability of human cerebromicrovascular endothelial cells in vitro and the murine BBB in vivo . (a) Incubation of hCMEC/D3 cell monolayers with pCS (10 µm, 100 µm, 1 mm; 24 h) dose-dependently increased paracellular permeability to a 70 kDa fitc-dextran conjugate; data are mean ± s.e.m., n = 9 independent experiments. (b) incubation of hCMEC/D3 cell monolayers with pCS (10 µm, 100 µm, 1 mm; 24 h) dose-dependently reduced TEER; data are mean ± s.e.m., n = 9 independent experiments. (c, d) confocal microscopic analysis of expression of C) AF488-phalloidin labeled actin filaments (white) or d) the tight junction component zona occludens-1 (ZO-1; white) in hCMEC/D3 cells following treatment for 24 h with 10 µm pCS, nuclei are counterstained with DAPI (blue), scale = 15 µm. Images are representative of at least three independent experiments. (e) treatment of male C57Bl/6 mice by i.P. injection of pCS (10 mg/kg) caused a time-dependent increase in extravasation of Evans blue tracer into the CNS parenchyma, reaching statistical significance at both 2 h and 6 h post administration; data are mean ± s.e.m., n = 5-6 animals. (f) exposure of male C57Bl/6 by s.C. implantation with osmotic mini-pumps releasing pCS at 7.5 µg/h for 28 days significantly enhanced extravasation of sodium fluorescein into the brain parenchyma; data are mean ± s.e.m., n = 7-10 animals. (g, h) confocal microscopic analysis of expression of G) claudin-5 or H) zonula occludens-1 (ZO-1) in the brains of male C57Bl/6 exposed by s.C. implantation with osmotic mini-pumps releasing pCS at 7.5 µg/h for 28 days, nuclei are counterstained with DAPI (blue), scale = 10 µm. Images are representative of 7-10 animals; (i) volcano plot showing the 380 significantly differentially expressed genes among the 16,988 mouse genes examined in this study ( n = 5 animals per group (treated, negative control)). Black data points: no significant change in expression; red dots, significantly ( p < 0.05, Benjamini-Hochberg) upregulated expression in the pCS group compared with the negative control; blue dots, significantly ( p < 0.05, Benjamini-Hochberg) downregulated expression in the pCS group compared with the negative control; n = 5 per group. (j) biological processes (above) and molecular functions (below) of genes found to be significantly downregulated ( n = 289) upon exposure of mice to pCS, based on enrichr p value ranking from gene ontology analysis. (k) topological analysis of the KEGG network resulting from mapping the 380 significantly ( p < 0.05, Benjamini-Hochberg) differentially expressed genes onto the 17 KEGG Mus musculus metabolic pathways listed in . Blue solid circles, genes whose expression is significantly downregulated in the pCS group compared with the control; red solid circles, genes whose expression is significantly upregulated in the pCS group compared with the control. Thick outline colors around the solid circles correspond to the molecular functions listed in the legend. Network summary statistics are available in table S3.

Article Snippet: Secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse 1:500, both Stratech Scientific, UK, AF488-conjugated goat anti-rabbit or anti-mouse 1:500, both Thermofisher Scientific, UK.

Techniques: Permeability, In Vitro, In Vivo, Incubation, Expressing, Labeling, Injection, Negative Control, Control

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 regulates cerebromicrovascular cell permeability in vitro through activation of the EGFR. (a) Stimulation of hCMEC/D3 cells with pCS (24 h, 10 µm) has no effect on cell surface expression of EGFR; data are mean ± s.e.m., n = 4 independent experiments; representative flow cytometry histograms are shown. (b) treatment of hCMEC/D3 cells with pCS (10 µm) causes a time-dependent increase in EGFR Tyr1068 phosphorylation, maintained for at least 30 min; data are mean ± s.e.m., n = 9 independent experiments; representative flow cytometry histograms are shown. (c) pre-treatment of hCMEC/D3 cells with erlotinib (2.5 µm, 10 min) prevents EGFR Tyr1068 phosphorylation in response to pCS treatment (10 µm, 30 min), data are mean ± s.e.m., n = 6 independent experiments; representative flow cytometry histograms are shown. (d) pre-treatment of hCMEC/D3 cell monolayers with erlotinib (2.5 µm, 10 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 = 9 independent experiments. (e) pre-treatment of hCMEC/D3 cell monolayers with erlotinib (2.5 µm, 10 min) prevents the reduction in TEER induced by pCS stimulation (10 µm, 24 h); data are mean ± s.e.m., n = 9 independent experiments. (f) 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), erlotinib (2.5 µm, 24 h) or both (erlotinib 2.5 µm, 10 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale bar = 15 µm. Images are representative of at least three independent experiments. (g) confocal microscopic analysis of expression of AF488-conjugated phalloidin labeled actin filaments (white) in hCMEC/D3 cells following treatment with either pCS (10 µm, 24 h), erlotinib (2.5 µm, 24 h) or both (erlotinib 2.5 µm, 10 min pre-treatment, followed by pCS 10 µm, 24 h); nuclei are counterstained with DAPI (blue), scale bar = 15 µm. Images are representative of at least three independent experiments. (h) paracellular permeability to a 70 kDa fitc-dextran conjugate of hCMEC/D3 cell monolayers bearing siRNA sequences targeting the EGFR (siRNA 5, siRNA 11, siRNA 12), a non-targeting negative control siRNA sequence, or that had been mock-transfected, with or without stimulation with pCS (10 µm, 24 h); data are mean ± s.e.m., n = 4 independent experiments. (i) TEER of hCMEC/D3 cell monolayers bearing siRNA sequences targeting the EGFR (siRNA 5, siRNA 11, siRNA 12), a non-targeting negative control siRNA sequence, or that had been mock-transfected, with or without stimulation with pCS (10 µm, 24 h); data are mean ± s.e.m., n = 3 independent experiments.

Article Snippet: Secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse 1:500, both Stratech Scientific, UK, AF488-conjugated goat anti-rabbit or anti-mouse 1:500, both Thermofisher Scientific, UK.

Techniques: Permeability, In Vitro, Activation Assay, Expressing, Flow Cytometry, Labeling, Negative Control, Sequencing, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

doi: 10.1186/s12964-024-01846-w

Figure Lengend Snippet: Detection of IL-22R1 expression on transfected HEK293T cells and binding of ABR variants to IL-22R1. Representative images of HEK293T cells expressing human IL-22R1 on the surface. Cells were incubated with biotinylated ABR variants (ABR006, ABR023, ABR027, ABR089, ABR099, ABR102, and ABR167) and stained with Streptavidin-AF568 (STV-AF568 conjugate) and anti-IL-22R1 antibody (anti-IL-22R1-AF488)

Article Snippet: After 1 h incubation, cells were washed, and goat anti-rabbit IgG conjugated with AF488 (Abcam, Cambridge, United Kingdom) was added for next 1 h staining in the dark.

Techniques: Expressing, Transfection, Binding Assay, Incubation, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

doi: 10.1186/s12964-024-01846-w

Figure Lengend Snippet: Fluorescence microscopy images demonstrating ABR variant’s binding to the IL-22R1 on stimulated HaCaT cells. HaCaT cells were stimulated with TNFα and IFNγ for 24 h. Double-staining of ABR proteins detected by Streptavidine-AF568 conjugate (red) and rabbit anti-IL-22R1 pAb-AF488 (green) was carried out

Article Snippet: After 1 h incubation, cells were washed, and goat anti-rabbit IgG conjugated with AF488 (Abcam, Cambridge, United Kingdom) was added for next 1 h staining in the dark.

Techniques: Fluorescence, Microscopy, Binding Assay, Double Staining