Journal:

Article Title: Expression of the Two Major Envelope Proteins of Equine Arteritis Virus as a Heterodimer Is Necessary for Induction of Neutralizing Antibodies in Mice Immunized with Recombinant Venezuelan Equine Encephalitis Virus Replicon Particles

doi:

Figure Lengend Snippet: Induction of neutralizing antibodies in mice with EAV-VRPs and whole EAV

Article Snippet: The neutralizing-antibody titers in these mice were comparable to or higher than those in mice repeatedly immunized with whole EAV (Table ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Immunogen Immunization schedule (weeks) Dose (IU a or TCID 50 b ) Weeks after primary immunization Neutralizing antibody titers c of mouse no.: 1 2 3 4 pVR21-G L VRP Primary (0) 6 × 10 5 3 0 0 0 0 First boost (3) 5 0 0 0 0 Second boost (7) 9 0 0 0 0 pVR21-M VRP Primary (0) 6 × 10 6 3 0 0 0 0 First boost (3) 5 0 0 0 0 Second boost (7) 9 0 0 0 0 pVR100-G L /M VRP Primary (0) 3.6 × 10 6 3 32 32 64 64 First boost (3) 5 512 512 512 512 Second boost (7) 9 2,048 1,024 2,048 2,048 EAV ATCC strain Primary (0) 2 × 10 4 b N/A d ND e ND ND ND First boost (4) N/A ND ND ND ND Second boost (7) 9 1,024 1,024 1,024 512 Open in a separate window a Infectious units as determined by immunoperoxidase staining of infected BHK-21 cells. b 50% tissue culture infectious doses. c Neutralization titers are expressed as the inverse of the antibody dilution providing 50% protection of RK-13 cell monolayers against 200 50% tissue culture infective doses of virus. d N/A, not applicable. e ND, not determined.

Techniques:

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: miR-503-5p inhibits proliferation, migration, and angiogenic abilities of HCAECs while promoting the proliferation and migration of HCASMCs. HCAECs and HCASMCs were treated with exogenous miR-503-5p mimic (with miR-mimic NC as a control) or exogenous miR-503-5p inhibitor (with miR-inhibitor NC as a control). ( A ) The expression of miR-503-5p (normalized to U6) in HCAECs and HCASMCs determined by RT-qPCR. ( B ) The proliferation of HCAECs and HCASMCs detected by CCK-8 assay. ( C ) The migration of HCAECs detected by transwell migration assays and scratch test. ( D ) Vessel-like tubes formed in HCAECs detected by Matrigel-based angiogenesis assays. ( E ) Apoptosis of HCAECs detected by flow cytometry. ( F ) The migration of HCASMCs detected by transwell migration assays and scratch test. Values obtained from three independent experiments in triplicate were analyzed by one-way ANOVA followed by Tukey's post hoc test among three or more groups. * p < 0.05 compared with HCAECs or HCASMCs treated with miR-mimic NC; * p < 0.05 compared with HCAECs or HCASMCs treated with miR-inhibitor NC.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Control, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: miR-503-5p negatively regulates expression of smad7 and smurf1/smurf2 and reduces the ubiquitination of TGF-β1 receptor. ( A ) Box plots of smad7 expression in human atherosclerosis-related microarray data GSE9490 consisted of 4 normal samples without DL-homocysteine and 8 DL-homocysteine-induced atherosclerosis samples. The blue box on the left indicates the smad7 expression of normal samples, while the red box on the right indicates the smad7 expression of diseased samples. ( B ) Box plots of smad7 expression in mouse atherosclerosis-related microarray data GSE2372. There were 3 samples obtained from the WT mice and 3 samples from ApoE mice with atherosclerosis. The blue box on the left indicates the smad7 expression in normal samples, while the red box on the right indicates the smad7 expression in diseased samples. ( C ) Venn map of predicted upstream miRNAs of smad7 in the DIANA TOOLS (miTG score > 0.90; http://diana.imis.athena-innovation.gr/DianaTools/index.php ), TargetScan (context++ score percentile ≥ 99; http://www.targetscan.org/vert_71/ ), miDIP (Integrated Score > 0.60; http://ophid.utoronto.ca/mirDIP/ ), miRWalk (|energy| ≥ 25; http://mirwalk.umm.uni-heidelberg.de/ ), microRNA, and starBase (pancancerNum ≥ 5; http://starbase.sysu.edu.cn/ ). ( D ) Prediction binding site of miR-503-5p in smad7, smurf1, and smurf2 3'-UTR analyzed by TargetScan software. ( E ) Detection of luciferase activity using dual-luciferase reporter gene assay. In panels ( F – H ), HCAECs were treated with exogenous miR-503-5p mimic (with miR-mimic NC as control) or exogenous miR-503-5p inhibitor (with miR-inhibitor NC as control). ( F ) The mRNA levels of TGF-β1, smad7, smurf1, and smurf2 (normalized to GAPDH) in HCAECs determined by RT-qPCR. ( G ) Protein levels of TGF-β1, smad7, smurf1, and smurf2 (normalized to GAPDH) in HCAECs determined by Western blot analysis. ( H ) Effects of Smad7 on the ubiquitination of TGF-β 1 by Smurf1 and Smurf2 detected by IP assay. Values obtained from three independent experiments in triplicate were analyzed by one-way ANOVA followed by Tukey's post hoc test among three or more groups. * p < 0.05 compared with HCAECs or HCASMCs treated with miR-mimic NC; * p < 0.05 compared with HCAECs or HCASMCs treated with miR-inhibitor NC.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Ubiquitin Proteomics, Microarray, Binding Assay, Software, Luciferase, Activity Assay, Reporter Gene Assay, Control, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: Delivery of miR-503-5p to HCAECs and HCASMCs by macrophage-derived EVs. ( A ) Structure and diameter of EVs observed by TEM (×100000). ( B ) Diameter and number of EVs measured by NTA. ( C ) Expression of EV marker proteins Alix, CD63, and CD9 determined by Western blot analysis; *p < 0.05 compared with EVs. HCAECs and HCASMCs were co-cultured with EVs derived from RAW264.7 cells with or without ox-LDL treatment. ( D ) Expression of miR-503-5p (normalized to U6), TGF-β1, smad7, smurf1, and smurf2 (all normalized to GAPDH) in HCAECs and HCASMCs determined RT-qPCR; *p < 0.05 compared with EVs derived from RAW264.7 cells without ox-LDL treatment. ( E ) EVs were phagocytosed by HCAECs and HCASMCs, observed under laser confocal microscope. PKH67-labeled EVs was green, DAPI-stained nuclei was blue, while cy3-miR-503-5p-labeled EVs was red (×400). ( F ) Protein expression of TGF-β1, smad7, smurf1, and smurf2 (normalized to GAPDH) in HCAECs determined by Western blot analysis. Values obtained from three independent experiments in triplicate were analyzed by unpaired t test between two groups and by one-way ANOVA followed by Tukey's post hoc test among three or more groups. *p < 0.05 compared with miR-inhibitor NC-treated HCAECs co-cultured with EVs in the absence of ox-LDL; # p < 0.05 compared with miR-503-5p inhibitor treated HCAECs co-cultured with EVs in the absence of ox-LDL.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Derivative Assay, Expressing, Marker, Western Blot, Cell Culture, Quantitative RT-PCR, Microscopy, Labeling, Staining

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: Macrophage-EVs carrying miR-503-5p inhibit proliferation, migration, and angiogenic abilities of HCAECs while promoting proliferation and migration of HCASMCs. HCAECs and HCASMCs treated with miR-503-5p inhibitor (with miR-503-5p in-NC as control) were co-cultured with EVs released by RAW264.7 cells with or without ox-LDL treatment. ( A ) Proliferation of HCAECs detected by CCK-8 assay. ( B ) Migration of HCAECs detected by transwell migration assays and scratch test. ( C ) Vessel like tubes formed in HCAECs detected by Matrigel-based angiogenesis assays; Scale bar = 20 μm. ( D ) Apoptosis of HCAECs detected by flow cytometry. ( E ) Expression of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) and adhesion molecules (ICAM-1 and VCAM-1) measured by ELISA methods. ( F ) Proliferation of HCASMCs detected by CCK-8 assay. ( G ) Migration of HCASMCs detected by transwell migration assays and scratch test. Values obtained from three independent experiments in triplicate were analyzed b y oneway ANOVA followed by Tukey's post hoc test among three or more groups. *p < 0.05 compared with miR-inhibitor NC-treated HCAECs co-cultured with EVs in the absence of ox-LDL; # p < 0.05 compared with miR-503-5p inhibitor-treated HCAECs co-cultured with EVs in the absence of ox-LDL.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Control, Cell Culture, CCK-8 Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: Overexpression of Smad7, smurf1, and smurf2 abrogates the effects of miR-503-5p on HCAECs and HCASMCs. HCAECs and HCASMCs were treated with exogenous miR-503-5p mimic (with miR-mimic NC as control) and expression vectors containing smad7, smurf1, or smurf2 gene. ( A ) Proliferation of HCAECs and HCASMCs detected by CCK-8 assay. ( B ) Migration of HCAECs detected by transwell migration assays and scratch test (×200). ( C ) Vessel-like tube-forming ability in HCAECs detected by Matrigel-based angiogenesis assays (×100). ( D ) Apoptosis of HCAECs detected by flow cytometry. ( E ) Migration of HCASMCs detected by transwell migration assays and scratch test (×200). Values obtained from three independent experiments in triplicate were analyzed by one-way ANOVA followed by Tukey's post hoc test among three or more groups. * p < 0.05 compared with HCAECs or HCASMCs treated with miR-mimic NC; # p < 0.05 compared with miR-503-5p mimic.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Control, Expressing, CCK-8 Assay, Migration, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis

doi: 10.18632/aging.103855

Figure Lengend Snippet: Systematic diagrams illustrating how macrophages crosstalk with ECs and SMCs and how they involve in the formation of atherosclerotic plaques. Macrophages deliver miR-503-5p to HCAECs and HCA-SMCs via EVs and downregulate Smad7, smurf1, and smurf2 in HCAECs and HCASMCs, thereby inhibiting proliferation, migration, and angiogenic abilities of HCAECs, but promoting proliferation and migration of HCASMCs.

Article Snippet: Human monocyte cell line THP-1, RAW264.7 mouse cell line (monocyte-macrophage leukemia cells), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration

Journal: Scientific Reports

Article Title: A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy

doi: 10.1038/s41598-017-00104-z

Figure Lengend Snippet: Macrophage content in carotid artery and aortic root atherosclerotic lesions. ( a ) Macrophage content in the carotid artery lesions as measured by MOMA2 staining was reduced upon treatment with 15a, both in total macrophage positive area (left panel) and relatively as percentage of lesion size (right panel). Micrographs show representative images of the individual groups (100X magnification). ( b ) Also in the aortic root, 15a significantly reduced the macrophage + area (left panel). Relative macrophage content did not significantly differ between the groups (right panel). Micrographs show representative images of the individual groups (40X magnification). *P < 0.05, **P < 0.01. Graphs shown are representative from one experiment with n = 10 controls versus n = 9 15a-treated mice.

Article Snippet: Macrophage content of the lesions in carotid arteries and aortic roots was stained using a rat monoclonal MOMA2 antibody (1:1000, Serotec, Kidlington, Oxford, UK).

Techniques: Staining