Journal: Cells

Article Title: miR-23a-3p is a Key Regulator of IL-17C-Induced Tumor Angiogenesis in Colorectal Cancer

doi: 10.3390/cells9061363

Figure Lengend Snippet: IL-17C stimulation promotes human intestinal microvascular endothelial cells (HIMEC) angiogenesis. ( A ) Representative bands of RT-PCR and quantitative analyses of two receptors of IL-17C, IL-17RA ( A , left ), and IL-17RE ( A , right ) in HIMECs treated with vehicle control or IL-17C (100 ng/mL, 3 h) are shown ( n = 3). GAPDH was used for the loading control. NS, not significant. ( B ) HIMECs were seeded into polymerized Matrigel and treated with IL-17C (100 ng/mL, 4 h) for in vitro tube formation assays ( B , left ). Scale bar, 100 μm. Total tube length of photos was measured using ImageJ v.1.47 software ( n = 6) ( B , right ). Error bars, SD. ** p < 0.01 vs. the control group. ( C ) The mouse aortic ring assay was conducted to investigate ex vivo angiogenic activity of IL-17C (100 ng/mL, 14 d) ( C , top ). Scale bar, 50 μm. For quantification, total sprouted tube vessels of 3 independent photos from each group were measured using ImageJ v.1.47 software ( C , bottom ). Error bars, SEM ( n = 3). *** p < 0.001 vs. the control group. ( D ) For the wound healing assays, cells were seeded in a culture plate, incubated with culture media with/without IL-17C (100 ng/mL), and scratched to create wounds. Photos were taken 24 h after wound creation ( D , left ). Scale bar, 50 μm. A view of representative fields from images of the wound healing assays, analyzed using ImageJ v.1.47 software ( D , right ). Error bars, SD ( n = 6). ** p < 0.01 vs. the control group. ( E ) F-actin organization in endothelial cells determined via rhodamine-phalloidin staining ( n = 3). Scale bar, 50 μm. ( F ) PCR microarray was performed to investigate expression levels of angiogenesis-related genes by IL-17C (100 ng/mL, 3 h) treatment in HIMECs ( n = 1). ( G ) The mRNA levels of CXCL3, IL-8, MMP-1, and MMP-10 were analyzed by qPCR at 3 h post IL-17C treatment. Error bars, SD ( n = 3). ** p < 0.01 and *** p < 0.001 vs. the control group.

Article Snippet: Briefly, the cells were then washed with PBS, fixed with 10% formalin (Sigma-Aldrich) for 15 min, and permeabilized with Triton X-100 (0.1%, Daejung, Gyeonggi-do, South Korea) for 5 min. After permeabilization, the cells were washed twice and stained for 15 min in the dark with rhodamine–phalloidin (100 nM/well, Cytoskeleton, Denver, CO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vitro, Software, Aortic Ring Assay, Ex Vivo, Activity Assay, Incubation, Staining, Microarray, Expressing

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Gene expressions of endothelial antigens upon TNFα (10 ng/ml) treatment on human umbilical artery and vein cells for 24 h. Data represent mean ± s.d.; n = 3 biological replicates; two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (B) Flow cytometry analysis of ACKR1 protein expression on artery and vein cells after 48 h of stimulation with TNFα (10 ng/ml). Data represent mean ± s.d.; n = 3 biological replicates; two-way ANOVA; **** P < 0.0001. (C) Expression profile of Ackr1 in vascular endothelial subtypes of various organs from a mouse single-cell transcriptome atlas (accession code: E-MTAB-8077 ). Data were generated with EC Atlas web-based visualization.

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Flow Cytometry, Expressing, Generated

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Scatterplot of the plasma levels of anti-ACKR1 autoantibodies in COVID-19 survivors and non-infected controls (mean ± s.e.m., Mann–Whitney test). Anti-ACKR1 concentrations were calculated based on normalized ratio over positive control of human immunoglobulin G (50 μg/ml) and presented as fold change to non-infected controls. (B) Heatmap depicting plasma concentrations of anti-ACKR1 autoantibodies, cytokines (quantified by Luminex multiplex assay), and enumeration of circulating endothelial cells in COVID-19 survivors and non-infected controls. Spearman’s correlation analysis revealed positive associations between anti-ACKR1 levels and the quantity of circulating endothelial cells, as well as each cytokine listed, with significant P -values indicated. Heatmap was generated using the MORPHEUS visualization software. (C) Spearman’s correlation analysis between anti-ACKR1 levels and reactive hyperemia index in individuals (n = 10) with endothelial dysfunctions (characterized by natural log-transformed reactive hyperemia index < 0.51). Spearman’s correlation coefficient r and P -values (two-tailed test) are indicated. (D) Kaplan-Meier plot shows the probability of primary vascular disease outcomes in patients without prior established cardiovascular diseases in a median follow-up period of 6.7 yr. Hazard ratio and 95% confidence intervals (CI) compare the time of blood collection to the first occurrence of vascular outcomes according to the presence of anti-ACKR1 autoantibodies (n = 16 undetectable anti-ACKR1, n = 10 anti-ACKR1 > 0 μg/ml).

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Infection, MANN-WHITNEY, Positive Control, Luminex, Multiplex Assay, Generated, Software, Transformation Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Scatterplot of the plasma levels of anti-ACKR1 autoantibodies in COVID-19 survivors and non-infected controls (mean ± s.e.m., Mann–Whitney test). Anti-ACKR1 levels were determined based on flow cytometry detection of bound anti-ACKR1 to K562 cells, a human erythroleukemic cell line ectopically overexpressing ACKR1. (B) ACKR1 (DARC) polymorphism, G125A (rs12075) was genotyped to identify individuals carrying FYA (G125) and FYB (125A) alleles, indicated under “Observed Frequencies.” “Expected frequencies” were extracted from literature as a comparison. (C) Spearman’s correlation analysis to assess the associations between the circulating levels of anti-ACKR1 autoantibodies and red blood cell count (n = 46).

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Infection, MANN-WHITNEY, Flow Cytometry, Comparison, Cell Counting

Journal: Life Science Alliance

Article Title: Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody

doi: 10.26508/lsa.202402598

Figure Lengend Snippet: (A) Experimental workflow of intervening autoantibody-antigen interactions to investigate the functional impact of anti-ACKR1 autoantibodies on human endothelial cells. The three-dimensional structure of ACKR1 was produced using I-TASSER server. (B) Flow cytometry analysis of the percentage of necrotic and late apoptotic endothelial cells after 24 h of experimental treatments. (C) Transendothelial electrical resistance assay to evaluate endothelial barrier permeability after experimental treatments. (D) Number of transmigrated PBMCs in an endothelial-immune cell co-culture transwell assay. (E) Degree of cytotoxicity was determined by quantifying lactate dehydrogenase activity in cell-free supernatants obtained from endothelial cells cultured with purified immunoglobulin G (IgG) and/or PBMCs. (F) Degree of antibody-dependent cellular cytotoxicity was determined from endothelial cells exposed to purified IgG and PBMCs. Purifie IgG was pre-incubated with and without blocking peptide or liposome ACKR1. For (B, C, D, E, F), data represent mean ± s.d.; one-way ANOVA; * P < 0.05, ** P < 0.01; *** P < 0.001, **** P < 0.0001; ns, non-significant. Data points represent biological replicates.

Article Snippet: We custom made a microarray-based autoantibody detection kit (RayBiotech) where a glass slide surface was spotted with purified human recombinant ACKR1 protein (Cat #TP304680; OriGene) that could be recognized by ACKR1 autoantibodies present in human plasma.

Techniques: Functional Assay, Produced, Flow Cytometry, Permeability, Co-Culture Assay, Transwell Assay, Activity Assay, Cell Culture, Purification, Incubation, Blocking Assay

Journal: Scientific Reports

Article Title: Magnetically-driven 2D cells organization on superparamagnetic micromagnets fabricated by laser direct writing

doi: 10.1038/s41598-020-73414-4

Figure Lengend Snippet: Scanning electron micrographs of 2D microarray fabricated by LDW via TPP: ( a ) Ormo squares; ( b ) Ormo/MNPs squares; ( c ) Ormo superposed with Ormo/MNPs squares forming the chessboard-like 2D microarray; ( d ) inset from ( a ); ( e ) inset from ( b ); ( f ) inset from ( c ) showing Ormo/MNPs square (left) and Ormo square (right); ( g ) Inset from ( c ) showing the intersection of 4 squares from the 2D microarray; DHM images of 2D microarray: ( h ) 3D reconstructed hologram; ( i ) phase shift 2D map at the intersection of 4 squares; ( j ) line profile of the height difference between adjacent Ormo/MNPs and Ormo squares along the green line from ( i ).

Article Snippet: Figure 2 Images of 2D microarray obtained by enhanced dark field microscopy using the Cytoviva 3D module (MNPs represented as yellow dots (false colors)): ( a ) top view; ( b ) tilted view; ( c – e ) EDX mapping of iron, carbon and oxygen.

Techniques: Microarray

Journal: Scientific Reports

Article Title: Magnetically-driven 2D cells organization on superparamagnetic micromagnets fabricated by laser direct writing

doi: 10.1038/s41598-020-73414-4

Figure Lengend Snippet: Images of 2D microarray obtained by enhanced dark field microscopy using the Cytoviva 3D module (MNPs represented as yellow dots (false colors)): ( a ) top view; ( b ) tilted view; ( c – e ) EDX mapping of iron, carbon and oxygen.

Article Snippet: Figure 2 Images of 2D microarray obtained by enhanced dark field microscopy using the Cytoviva 3D module (MNPs represented as yellow dots (false colors)): ( a ) top view; ( b ) tilted view; ( c – e ) EDX mapping of iron, carbon and oxygen.

Techniques: Microarray, Microscopy

Journal: Scientific Reports

Article Title: Magnetically-driven 2D cells organization on superparamagnetic micromagnets fabricated by laser direct writing

doi: 10.1038/s41598-020-73414-4

Figure Lengend Snippet: 2D microarrays: ( a , f ) Optical images; ( b , g ) Green autofluorescence; Representative fluorescence microscope images of fibroblasts seeded on 2D microarrays: ( c , h ) cytoskeleton (red) visualized by staining with F-actin; ( d , i ) nuclei (blue) visualized by staining with Hoechst; ( e , j ) overlapped fluorescence images of 2D microarrays, cells cytoskeleton and nuclei; ( k , m ) 3D top and ( l , n ) 3D tilted views of cells nuclei (blue), cytoskeleton (red) and centers of MNPs and agglomerations of MNPs (yellow), imaged by enhanced dark field microscopy. The samples were imaged in the presence ( a – e , k , l ) and absence ( f – j , m , n ) of SMF. All cells were imaged after 1 day of cell culture.

Article Snippet: Figure 2 Images of 2D microarray obtained by enhanced dark field microscopy using the Cytoviva 3D module (MNPs represented as yellow dots (false colors)): ( a ) top view; ( b ) tilted view; ( c – e ) EDX mapping of iron, carbon and oxygen.

Techniques: Fluorescence, Microscopy, Staining, Cell Culture

Journal: Developmental cell

Article Title: YAP/TAZ and Hedgehog coordinate growth and patterning in gastrointestinal mesenchyme

doi: 10.1016/j.devcel.2017.08.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HRP conjugated donkey anti-rabbit , Jackson Immunoresearch , 711-035-152.

Techniques: Recombinant, Plasmid Preparation, Reporter Assay, Reverse Transcription, Microarray, Sequencing, Software