Journal: Aging and Disease

Article Title: Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction

doi: 10.14336/AD.2022.0316

Figure Lengend Snippet: Changes in eNOS, p53, p21, p16, cGAS, STING and p-IRF3/IRF3 expression levels in D-GAL-treated HAECs. (A) The eNOS, p53, p21, p16, cGAS, STING, IRF3, p-IRF3 and β-actin protein expression levels were measured in HAECs treated with 0, 5, 10, 15, 20 and 25 g/L D-GAL for 24 h by western blot analysis. The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (C). Data analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the 0 g/L group. n=6, * P <0.05 compared with the 0 g/L group. A concentration of 20 g/L was chosen for use in subsequent experiments. (B) The eNOS, p53, p21, p16, cGAS, STING, IRF3, p-IRF3 and β-actin protein expression levels were measured in HAECs at 0, 6, 12, 24 and 48 h after treatment with 20 g/L D-GAL by western blot analysis. The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (D). Data were analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the 0 h group. n=6, * P <0.05 compared with the 0 h group. A time point of 48 h was chosen for use in subsequent experiments

Article Snippet: RU.521 (Cat# HY-114180), H-151 (Cat# HY-112693) and D-GAL (Cat# HY-N0210) were obtained from MCE (NJ, USA).

Techniques: Expressing, Western Blot, Concentration Assay

Journal: Aging and Disease

Article Title: Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction

doi: 10.14336/AD.2022.0316

Figure Lengend Snippet: Effects of RU.521 on eNOS, cell senescence, inflammatory cytokines and NO production in D-GAL-treated HAECs. 3μM RU.521 was used to treat the HAECs. eNOS, p53, p21, p16, cGAS, p-STING, STING, IRF3, p-IRF3 and β-actin expression levels in HAECs from the Control, D-GAL and D-GAL+RU.521 groups were examined by western blot analysis (A). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (B). mRNA expression of IFNβ (C), Ifit1 (D), Ifit2 (E), Ifit3 (F), MCP-1 (G), IL-1β (H), IL-6 (I) and TNF-α (J) in HAECs from the Control, D-GAL and D-GAL+RU.521 groups was determined by PCR. NO release into the culture medium of HAECs from the Control, D-GAL and D-GAL+RU.521 groups was measured (K). Representative photomicrographs and quantitative analysis of SA-β-gal-positive staining in the Control, D-GAL and D-GAL+RU.521 groups (L and M). Data were analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the Control group. n=6, * P <0.05 compared with the Control group, # P <0.05 compared with the D-GAL group.

Article Snippet: RU.521 (Cat# HY-114180), H-151 (Cat# HY-112693) and D-GAL (Cat# HY-N0210) were obtained from MCE (NJ, USA).

Techniques: Expressing, Control, Western Blot, Staining

Journal: Aging and Disease

Article Title: Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction

doi: 10.14336/AD.2022.0316

Figure Lengend Snippet: Effects of siRNA-cGAS on eNOS, cell senescence, inflammatory cytokines and NO production in D-GAL-treated HAECs. cGAS expression in the Control, Negative Control (NC) and si-cGAS groups was measured by western blot analysis 48 h after transfection (A). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (B). Data were analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. n=6, & P <0.05. eNOS, p53, p21, p16, cGAS, p-STING, STING, IRF3, p-IRF3 and β-actin expression levels in HAECs from the Control, D-GAL and D-GAL+si-cGAS groups were examined by western blot analysis (C). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (D). mRNA expression of IFNβ (E), Ifit1 (F), Ifit2 (G), Ifit3 (H), MCP-1 (I), IL-1β (J), IL-6 (K) and TNF-α (L) in HAECs from the Control, D-GAL and D-GAL+si-cGAS groups was determined by PCR. NO release into the culture medium of HAECs from the Control, D-GAL and D-GAL+si-cGAS groups was measured (M). Representative photomicrographs and quantitative analysis of SA-β-gal-positive staining in the Control, D-GAL and D-GAL+si-cGAS groups (N and O). Data were analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the Control group. n=6, * P <0.05 compared with the Control group, # P <0.05 compared with the D-GAL group.

Article Snippet: RU.521 (Cat# HY-114180), H-151 (Cat# HY-112693) and D-GAL (Cat# HY-N0210) were obtained from MCE (NJ, USA).

Techniques: Expressing, Control, Negative Control, Western Blot, Transfection, Staining

Journal: Aging and Disease

Article Title: Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction

doi: 10.14336/AD.2022.0316

Figure Lengend Snippet: Effects of H-151 on eNOS, cell senescence, inflammatory cytokines and NO production in D-GAL-treated HAECs. H-151 (3 μM) was used to treat HAECs. eNOS, p53, p21, p16, cGAS, STING, IRF3, p-IRF3 and β-actin expression levels in HAECs from the Control, D-GAL and D-GAL+H-151 groups were examined by western blot analysis (A). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (B). mRNA expression of IFNβ (C), Ifit1 (D), Ifit2 (E), Ifit3 (F), MCP-1 (G), IL-1β (H), IL-6 (I) and TNF-α (J) in HAECs from the Control, D-GAL and D-GAL+H-151 groups was determined by PCR. NO release into the culture medium of HAECs from the Control, D-GAL and D-GAL+H-151 groups was measured (K). Representative photomicrographs and quantitative analysis of SA-β-gal-positive staining in the Control, D-GAL and D-GAL+H-151 groups (L and M). Data were analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the Control group. n=6, * P <0.05 compared with the Control group, # P <0.05 compared with the D-GAL group.

Article Snippet: RU.521 (Cat# HY-114180), H-151 (Cat# HY-112693) and D-GAL (Cat# HY-N0210) were obtained from MCE (NJ, USA).

Techniques: Expressing, Control, Western Blot, Staining

Journal: Aging and Disease

Article Title: Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction

doi: 10.14336/AD.2022.0316

Figure Lengend Snippet: Effects of si-STING on eNOS, cell senescence, inflammatory cytokines and NO production in D-GAL-treated HAECs. STING expression in the Control, Negative Control (NC) and si-STING groups was measured 48 h after transfection (A). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (B). Data analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. n=6, & P <0.05. eNOS, p53, p21, p16, STING, IRF3, p-IRF3 and β-actin protein expression levels in HAECs from the Control, D-GAL and D-GAL+si-STING groups were examined by western blot analysis (C). The β-actin was used as the housekeeper protein for normalization. Quantification of the protein levels is shown in (D). mRNA expression of IFNβ (E), Ifit1 (F), Ifit2 (G), Ifit3 (H), MCP-1 (I), IL-1β (J), IL-6 (K) and TNF-α (L) in HAECs from the Control, D-GAL and D-GAL+si-STING groups was assessed by PCR. NO release in the medium of the Control, D-GAL and D-GAL+si-STING groups was measured (M). Representative photomicrographs and quantitative analysis of SA-β-gal-positive staining in the Control, D-GAL and D-GAL+si-STING groups (N and O). Data analyzed by one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the Control group. n=6, * P <0.05 compared with the Control group, # P <0.05 compared with the D-GAL group.

Article Snippet: RU.521 (Cat# HY-114180), H-151 (Cat# HY-112693) and D-GAL (Cat# HY-N0210) were obtained from MCE (NJ, USA).

Techniques: Expressing, Control, Negative Control, Transfection, Western Blot, Staining

Journal: Biomolecules

Article Title: The Ensemble of Conformations of Antifreeze Glycoproteins (AFGP8): A Study Using Nuclear Magnetic Resonance Spectroscopy

doi: 10.3390/biom9060235

Figure Lengend Snippet: Sequence-specific chemical shift assignments of antifreeze glycoprotein fraction 8 (AFGP8). The 2D TOCSY NMR spectra of AFGP8-BS (AFGP8 from Boreogadus saida ) ( a ) and AFGP8-TB (AFGP8 from Trematomus borchgrevinki ) ( b ), showing the amide to alpha and methyl proton regions. The TOCSY patterns associated with all the amino acid residues of the primary sequence, except for the alanine residue at position 1 and proline residues at positions 4 and 10 in AFGP8-BS and positions 7 and 13 in AFGP8-TB.

Article Snippet: The disaccharide beta- d -galactosyl-(1,3)-alpha-N-acetyl- d -galactosamine (5 mg, 98% purity, CAS 3554-90-3, Cat No. A152000) was purchased from Toronto Research Chemicals (North York, ON, Canada) and was used as purchased.

Techniques: Sequencing, Residue

Journal: Process Biochemistry

Article Title: A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB – Gene cloning, purification and characterization

doi: 10.1016/j.procbio.2014.09.018

Figure Lengend Snippet: Fig. 7. TLC of products of alkyl glycosides synthesis catalyzed by Arthrobacter sp. 32cB -d-galactosidase. Reaction mixtures containing lactose and 1-butanol (A), 2-propanol (B), 1-hexanol (C) or cyclohexanol (D) as substrates, and 2 U mL−1 Arthrobacter sp. 32cB -d-galactosidase were incubated at 30 ◦C for 8 h. Lane 1 – d-galactose, lane 2 – d-glucose,

Article Snippet: Materials Isopropyl- -d-thiogalactopyranoside (IPTG) and 5-bromo4-chloro-3-indolyl- -d-galactopyranoside (X-gal) were purchased from Biosynth AG (Switzerland). o-Nitrophenyl-d-galactopyranoside (ONPG), p-nitrophenyl- -d-galactopyranoside (PNPG), p-nitrophenyl- -d-glucopyranoside, p-nitrophenyl- -d-fucopyranoside, p-nitrophenyl- -d-xylopyranoside, p-nitrophenyl- -d-glucouronide, p-nitrophenyl- -d-manno pyranoside, p-nitrophenyl- -d-cellobioside, p-nitrophenyl- - l-arabinopyranoside, p-nitrophenyl- -d-galactopyranoside, p-nitrophenyl- -d-glucopyranoside, l-arabinose, d-xylose, lactulose, dithiothreitol (DTT), ethylenediaminetetraacetic acid sodium salt (EDTA), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) and tris(2-carboxyethyl)phosphine (TCEP) were supplied by Sigma (USA).

Techniques: Incubation

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: Influence of P-D2-EVs on Fut1-mediated fucosylation of ICAM1. A Western blot analysis of UEA-1-enriched mDCs under different treatments. B Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. C IP-based detection and quantification of ICAM1 binding to UEA1 in each group. D Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. E Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. F IP-based detection and quantification of ICAM1 binding to UEA1 in each group. G Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. H Schematic representation of Fut1-OE-iDCs treated with 2DGal; (I) Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. J IP-based detection and quantification of ICAM1 binding to UEA1 in each group. K Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm; UEA1: Ulex europaeus agglutinin 1, 2DGal: 2-deoxy- d -galactose. * indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Article Snippet: In the mDCs + 2DGal group, 2-deoxy-D-galactose (10 mM; MCE, HY-131892) was additionally induced from day 5 to 7.

Techniques: Western Blot, Binding Assay, Immunofluorescence, Staining, Control

Journal: Journal of Nanobiotechnology

Article Title: New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

doi: 10.1186/s12951-024-02748-2

Figure Lengend Snippet: Influence of P-D2-EVs on DC metabolism via the Fut1/ICAM1/P38 MAPK pathway. A , B Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. C mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. D Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. E Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry. F Schematic representation of the culture conditions for mDCs, mDCs + 2DGal, and m + 2DGal + Anisomycin groups. G Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. H mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. I Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. J Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry; 2DGal: 2-deoxy- d -galactose; *indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Article Snippet: In the mDCs + 2DGal group, 2-deoxy-D-galactose (10 mM; MCE, HY-131892) was additionally induced from day 5 to 7.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

doi: 10.1371/journal.pone.0086736

Figure Lengend Snippet: Structural overlay of the active sites in Tm P2O V546C (beige) and H450G (green). The V546C mutant is used as a reference since it displays the same binding modes as the H167A mutant, and for 2- or 3-fluorinated glucose, also agrees with the binding modes observed for the wild type. (a) Binding of 3F Glc in the productive 2-oxidation binding mode. The sugar is stabilized as the β -anomer with O2 coordinated by His458 and Asn593 and C2 appropriately positioned for oxidation. The substrate-binding loop is in the semi-open conformation positioning Phe454 closely packed against the pyranose as has been described for the productive binding mode earlier . (b) 2F Glc in the competing 3-oxidation binding mode. In H450G, the C1 hydroxyl in 2F Glc is stabilized in axial configuration ( α -anomer) by Asp452 and Thr169 and the substrate-binding loop assumes the semi-open conformation. V546C stabilizes the β -anomer and reveals the open conformation of the substrate-binding loop as observed earlier for H167A in complex with 2F Glc . (c) 3F Gal in productive 2-oxidation binding mode with the axial C4 hydroxyl group coordinated by Asp452 and Thr169. The substrate-binding loop is in the semi-open conformation. (d) 2F Gal in competing binding modes. The competing binding mode observed for V546C corresponds to the 2F Gal β -anomer oriented for oxidation at C1. The competing binding mode for H450G shows the α- anomer of 2F Gal oriented for oxidation at C3. In both cases, the substrate-binding loop assumes the semi-open conformation compatible with the productive sugar-oxidation mode. All structures that bind sugar substrate show the Thr169 Oγ1 atom pointing away from the flavin N(5)/O(4) locus, which constitutes an additional hallmark of the productive binding mode. For clarity, the covalent link between the flavin and His167 is not shown in the pictures. The pictures were produced using the program PyMOL .

Article Snippet: LLC., USA; Cat. N o . F5006-25 mg); 3F Gal (3-deoxy-3-fluoro-D-galactose; Carbosynth Ltd., Newbury, UK; Cat. N o . MD05336); and 2F Gal (2-deoxy-2-fluoro-D-galactose; Toronto Research Chemicals Inc.; Cat. N o . D233000).

Techniques: Mutagenesis, Binding Assay, Produced

Journal: PLoS ONE

Article Title: Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

doi: 10.1371/journal.pone.0086736

Figure Lengend Snippet: Superposition of mutant structures emphasizing the conformation of the substrate-binding loop. The FAD molecule and the pyranose sugar are shown as ball-and-stick models. (a) Tm P2O variants complexed with 3F Glc corresponding to the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The relaxation induced by the H450G replacement (green and light-blue models) is highlighted by a shaded circle. The H167A model corresponds to PDB code 3PL8 . (b) Mutant complexes with bound 2F Glc in the competing 3-oxidation binding mode. The H167A (PDB code 2IGO ) and V546C variants show the open loop conformation, which also has been observed for the wild type. H450G and H450G/V546C show the productive semi-open loop conformation. (c) Mutant complexes of V546C, H450G and H450G/V546C with bound 3F Gal in the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The wild-type mimic H167A displays did not bind the sugar and displays the closed, occluded loop conformation that is typically observed for Tm P2O in the absence of oxidizable sugar. The closed loop conformation is incompatible with sugar binding. (d) Mutant complexes with bound 2F Gal. Despite the fundamentally different competing modes observed for H167A and V546C (C1-oxdiation mode) and H450G and H450G/V546C (C3-oxidation mode), all complexes show the substrate-binding loop in the semi-open loop conformation associated with productive sugar binding. The pictures were produced using the program PyMOL .

Article Snippet: LLC., USA; Cat. N o . F5006-25 mg); 3F Gal (3-deoxy-3-fluoro-D-galactose; Carbosynth Ltd., Newbury, UK; Cat. N o . MD05336); and 2F Gal (2-deoxy-2-fluoro-D-galactose; Toronto Research Chemicals Inc.; Cat. N o . D233000).

Techniques: Mutagenesis, Binding Assay, Produced