Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) The serum Hsp20 level was increased in response to in vivo 30 min-LAD occlusion followed by 24 h-reperfusion. Cardiac-specific overexpression of Hsp20 increased the Hsp20 concentration in the serum under basal and myocardial ischemia/reperfusion conditions. (n = 6; *, p<0.05 vs. WTs; #, p<0.05 vs. Sham groups). (B) The levels of Hsp20 in hearts from Hsp20-transgenic mice were determined by Western blot. α-Actin was used as an internal control (n = 4). (C) Myocardial ischemia/reperfusion stimulated the translocation of Hsp20 to the cardiomyocyte membrane, which was detected by fluorescence microscopy. Images are representative sections from four mice per group (green, Hsp20; red, α-Actin; Scale bar, 100 µm). (D) Quantitative data for expression of Hsp20 was evaluated using IPP 5.1 (n = 4; *, p<0.05 vs. WTs; #, p<0.05 vs. Sham groups).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: In Vivo, Over Expression, Concentration Assay, Transgenic Assay, Western Blot, Control, Translocation Assay, Membrane, Fluorescence, Microscopy, Expressing

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Mild stress dose-dependently increased the amount of Hsp20 released from both Ad.GFP and Ad.Hsp20 infected myocytes, and (B) there was no difference in the release of lactate dehydrogenase (LDH), a marker of necrosis. Similar results were observed in two additional, independent experiments (*, p<0.05 vs. Ad.GFP-Control; #, p<0.05 vs. Ad.GFP).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Infection, Marker, Control

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Brefeldin A (BFA), which inhibits the classical protein transport pathway, did not block Hsp20 release into the media under either basal or hypoxia conditions (20 µM H 2 O 2 ). However, the release of Hsp20 from cardiomyocytes was reduced by both dimethyl amiloride (DMA), an exosome inhibitor, and Methyl-β-cyclodextrin (MBC), an inhibitor of lipid raft formation via depletion of membrane cholesterol. (B) The activity of acetylcholine esterase was used to quantify the amount of exosomes present in the media after various treatments. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Basal-Control; #, p<0.05 vs. H 2 O 2 -Control).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Blocking Assay, Membrane, Activity Assay, Control

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Recombinant human Hsp20 protein was added to HUVECs at various doses (80–2000 ng/ml) for 24 h. BSA was used as a control. Cell proliferation was determined by MTS. (B) Time-course effects of the Hsp20 protein(1000 ng/ml) on the HUVEC proliferation. (C) Representative photographs indicated the effects of recombinant human Hsp20 protein on the trans-well and tube formation of HUVECs. (D) Migration was quantified by counting cells that were moved through the membrane (Trans-well assay). (E) Tube formation was evaluated by the measurement of relative tube length. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Control).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Recombinant, Control, Migration, Membrane

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) VEGFR2 coated on a plate dose-dependently captured the Hsp20 protein, whereas BSA coated did not arrest the Hsp20 protein. (B) Recombinant human Hsp20 protein significantly induced the expression of VRGFR2 in HUVECs, and co-localized with VEGFR2 in the cell surface. Images are representative sections from 20 fields per group (green, Hsp20; red, VEGFR2). Scale bar, 25 µm. (C–E) Blockade of the VEGFR2 signaling by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor) suppressed the HUVEC migration (C and D) and tube formation (C and E). Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Control). (F and G) Immunoblots determined the levels of Akt, p-Akt, ERK and p-ERK in Hsp20-treated HUVECs. IgG or VEGFR2 antibody was pre-treated 30 min prior to the addition of Hsp20. β-actin was used as an internal control (n = 4; *, p<0.05 vs. Basal; #, p<0.05 vs. IgG+Hsp20).

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Recombinant, Expressing, Migration, Control, Western Blot

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: (A) Blood vessels were stained for CD31 (capillary density) in heart sections of WT and Hsp20 TG mice, and (B) their quantitative analysis. For quantification of positively stained vessels, five sections of each heart (n = 4 hearts per group) were analyzed by an investigator who was blinded with respect to samples. Blood vessels were detected at low magnification (×200). Images are representative sections from four mice per group (green, α-Actin; red, CD31). Scale bar, 50 µm.

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Staining

Journal: PLoS ONE

Article Title: Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

doi: 10.1371/journal.pone.0032765

Figure Lengend Snippet: Intracellular Hsp20 is released outside cardiomyocytes via exosomes, and then interacts with VEGFR2. Consequently, its downstream signaling pathways (i.e. Akt and ERK) are activated, which promote myocardial angiogenesis.

Article Snippet: Samples or recombinant mouse Hsp20 protein (EIAab Science) were added and incubated for 2 h followed by three washes in PBST.

Techniques: Protein-Protein interactions

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Science Advances

Article Title: A20 haploinsufficiency disturbs immune homeostasis and drives the transformation of lymphocytes with permissive antigen receptors

doi: 10.1126/sciadv.adl3975

Figure Lengend Snippet: κ ( A ) Immunoblotting of A20 and NF-κB p65 in whole-cell extract from B cell lymphoma cell lines. ( B ) Immunoblotting of NF-κB p65 in cytosolic and nuclear fractions of indicated cell lines. TBP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading controls. ( C ) Quantification of mean (±SD) TNF secretion by indicated cell lines ( n = 3 to 4 biological replicates). ( D ) Mean (±SD) proliferation and ( E ) viability of indicated cell lines ( n = 6 biological replicates) after 72 hours of incubation with anti-TNF antibody infliximab or human IgG (Intratect). Dotted lines represent seeded cell number. ( F ) Immunoblotting of A20 and NF-κB p65 after lentiviral transduction of A20 in RI-1 cells. ( G ) Immunoblotting of NF-κB p65 in cytosolic and nuclear fractions after A20 reconstitution in RI-1 cells. ( H ) Mean (±SD) TNF secretion after lentiviral transduction of A20 in RI-1 cells ( n = 3 biological replicates). ( I ) Mean (±SD) proliferation of A20-overexpressing RI-1 cells in the presence of infliximab ( n = 4 biological replicates). Statistics: one-way ANOVA. ( J ) Volcano plot showing differential gene expression between RI-1 wt and RI-1 + pLeGO-A20 as determined by RNA-seq performed in duplicates. Genes were considered differentially expressed with an adjust P < 0.01 and log 2 fold change >1. ( K ) Top down-regulated signaling pathways in RI-1 + pLeGO-A20 cells. All adjusted P values <0.0001. ( L ) Immunoblotting of NF-κB p65 in whole-cell extract and quantification of secreted TNF after incubation with 100 μM PDTC for 1 hour ( n = 3 biological replicates). ( M ) Quantification of mean (±SD) TNF secretion after stimulation with 100 ng/ml recombinant TNF for 1 hour ( n = 3 biological replicates). * P < 0.05; ** P < 0.01.

Article Snippet: For inhibition or activation of NF-κB, 2 × 10 6 cells were seeded in six-well plates and incubated with 100 μM PDTC ammonium (Selleckchem) or recombinant TNF (100 ng/ml, Sigma-Aldrich) for 1 hour.

Techniques: Western Blot, Incubation, Transduction, Gene Expression, RNA Sequencing, Protein-Protein interactions, Recombinant