Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Diagram depicting the culture model used to generate time course samples from healthy human CD4+ memory T cells. Cells were stimulated with CD3/CD28 Dynabeads for 12 days, with fresh beads plus full media change every other day (fresh media alone added on alternate days). Samples were collected on days highlighted in bold. (B) Multiomics workflow (see for details). Experiments were performed in biological triplicate, with proteomics, phosphoproteomics, and metabolomics done on same samples. (C) ELISA-based quantification of secreted cytokines (IL-2, IFN-γ, TNF-α) on days 0, 6, 10, and 12, 3 replicates per time point. (D) Cell counts were taken each time point, and average cell division compared to previous time point calculated. Cell viability remained stable throughout at ~75%–85%. (E) Flow cytometric analysis of inhibitory receptors PD-1, TIM-3, and TIGIT at resting, day 6, and day 12 time points. Data normalized to mode for accurate population comparisons to account for differences in number of cells analyzed. The data underlying can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: Mean fluorescence intensity of PD-1 in CD4+ T cells from both HIV+ and HIV- patients (grouped due to observing same effect in both) for CD4+ cells that are CD276+ or FLT-1+. The data underlying this figure can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Fluorescence, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Causal network analysis leveraging annotated interactions (transcription factor-target, kinase-substrate, phosphatase-substrate) as a framework to evaluate protein expression changes (late exhaustion (day 12) versus resting, taking into account previous time point comparisons) to predict key nodes (regulators) impacting most changes seen across the data set. (B) Pruned version of a large subnetwork with p300 (black outline) at main hub. Orange circles mark proteins of interest mentioned in main text. (C) Depiction of experimental design of p300 inhibition studies. (D) Flow cytometric analysis of PD-1 and TIM-3 showing shifts in fluorescent intensity of cell populations in treated versus untreated (cell number normalized to mode). Cell numbers shown for each plot are 20,312 for p300 treated and 30,438 for DMSO, which corresponds to all CD4+ T cells from the sample (excluding doublets and debris, see for gating), corresponding to >95% of sample. (E) Bar chart of mean fluorescent intensity showing significant changes ( T test) in PD-1 and TIM-3 during p300 inhibition versus controls (antigen removed for 2 days, or treatment with T-cell activation inhibitor Dasatinib); *, p -value <0.05, ***, p -value <0.005. (F) Heatmap showing recruitment of cofactors (p300, class I HDACs, KAT2B) to transcription factor family DNA motifs as measured using CASCADE protein-binding microarrays. The data underlying this figure can be found in and Data (A, B), supplemental flow cytometry files uploaded to flowrepositroy.org (D), and the (E, F). Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Expressing, Inhibition, Activation Assay, Protein Binding, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: UMAPs of CD4+ subtypes and CD8+ T exhausted tumor lymphocytes showing gene expression of (A) exhaustion-associated genes, and (B) gene expression of proteins identified in the model presented by Lawton and colleagues. UMAPs made using Single Cell Portal, Tarhan and colleagues. The data from this figure and tool used to make it can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP1039/a-single-cell-and-spatially-resolved-atlas-of-human-breast-cancers . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Gene Expression

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza modulated the neutrophil-associated TIME in the lungs. (A) Experimental setup. The mice were constructed as experimental metastasis models after a period of acclimatization. The mice were prepared as experimental metastasis models following an acclimatization period. Ginseng and Salvia miltiorrhiza extracts were administered daily to the relevant groups according to the schematic diagram, and lung tissues were collected on day 28. (B) Representative bioluminescence images of metastasis in the intravenously implanted LLC mice. (C) Representative H&E images of the tumor area in the lungs of the mice (scale bar: 50 μm). (D) Quantitative analysis of the lung tumor area ( n = 3). (E) Quantitative analysis of the number of lung metastatic nodules in tumor-bearing mice( n = 15). (F) Metastasis rates of LLC tumor-bearing mice ( n = 15). (G) GOBP analysis of intersecting genes in the control, model and G + S groups. (H) GSEA of genes related to neutrophil migration. (I) Representative images of immunohistochemical staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 50 μm). (J) Representative images of immunofluorescence staining and quantitative analysis of Ly6G in the lungs ( n = 3; scale bar: 100 μm). All the data are presented as mean ± SEM. #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Construct, Control, Migration, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: CPT inhibited CD62E–mediated neutrophil adhesion and recruitment to metastases. (A) Representative images and quantification of HL–60 cell adhesion to HUVECs (scale bar: 200 μm). (B) Schematic diagram of the parallel plate flow chamber combined with the microscope. (C) Number of neutrophils firmly adhered to HUVECs. (D) Representative immunofluorescence images and quantification of CD62E in the lungs ( n = 3; scale bar: 50 μm). (E) Representative western blot images of CD62E levels in the lungs and quantitative data ( n = 3). GAPDH was used as a loading control. (F) The mRNA expression levels of TNF–α, ICAM1, and VCAM1 in lung tissues were determined by qRT–PCR ( n = 3). (G) The correlation between CD62E expression and neutrophil infiltration in LUAD and LUSC was analyzed via TIMER. (H) Representative images and quantification of Ly6G + neutrophils in lungs detected by flow cytometry ( n = 4). (I) Representative immunofluorescence images and quantification of Ly6G + neutrophils in the lungs (n = 3; scale bar: 50 μm). All the data are presented as mean ± SEM. ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Microscopy, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Neutrophil RNA sequencing analysis and correlation analysis of NETs and lung cancer. (A) Schematic diagram of neutrophil isolation and RNA sequencing from mouse lung tissues. (B-C) Heatmap analysis of neutrophil-related genes and KEGG analysis of altered genes. (D) Results of partial GOBP analysis of intersecting genes in the control, model and G + S groups. (E) The expression of HIST3H3 in different cancers and adjacent normal tissues was analyzed via TIMER. (F–H) The correlation between copy number alterations in HIST3H3, ELANE, and MPO and neutrophil infiltration was analyzed via TIMER. (I) The correlations between cumulative survival and HIST3H3, ELANE, MPO and neutrophil infiltration in lung cancer patients were analyzed via TIMER. A two-sided Wilcoxon rank-sum test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: RNA Sequencing, Isolation, Control, Expressing

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Ginseng and Salvia miltiorrhiza reduced the generation of NETs in lung tissues, and effective substances from ginseng were screened. (A) Representative immunohistochemical images of MPO expression in the lungs (scale bar: 100 μm). (B) Representative immunofluorescence images of H3cit and NE in the lungs (scale bar: 100 μm). (C–E) Quantitative analysis of MPO, H3cit and NE expression in the lungs ( n = 3). (F) Neutrophil MPO release was detected by ELISA. The chemical structure of Rg1 is shown on the right. (G) Representative images and quantification of extracellular DNA release after Sytox™ Green staining (scale bar: 50 μm). All the data are presented as mean ± SEM. ####p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reduced NET formation by inhibiting ROS production and ERK1/2 and MAPK phosphorylation. (A) Representative immunofluorescence images of the NET components H3cit and NE in neutrophils ( n = 3; scale bar: 50 μm). (B–C) Representative western blot images of H3cit expression in neutrophils and quantitative data. GAPDH was used as a loading control ( n = 3). (D-E) ROS production in dHL-60 cells (D) and neutrophils (E) was assessed by flow cytometry. The median fluorescence intensity of ROS was used for quantification. (F-G) Representative western blot images and quantification of p-ERK1/2, ERK1/2, p-MAPK and MAPK expression in dHL-60 cells (F) and neutrophils (G). GAPDH was used as a loading control. All the data are presented as mean ± SEM. # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Synergistic potentiation of the anti-metastatic effect of a Ginseng - Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NETformation

doi: 10.1016/j.jare.2024.10.036

Figure Lengend Snippet: Rg1 reversed NET-induced invasion, EMT, and adhesion of LLC cells and increased vascular permeability. (A) LLC cell proliferation was detected via a CCK8 assay. (B) Schematic diagram of NET isolation and coculture with LLC cells in the transwell system. (C) Images of the transwell invasion assay (scale bar, 200 μm). The number of infiltrated cells and the OD values were used for quantification. (D-E) Representative immunofluorescence images and quantification of MMP9 in the lungs (n = 3, scale bar 50 μm). (F) Representative immunofluorescence images of E-cadherin and N-cadherin in LLC cells treated with or without Rg1 or NETs. (G-I) Representative immunofluorescence images and quantification of E-cadherin and N-cadherin in the lungs ( n = 3, scale bar 50 μm). (J) Schematic diagram of the adhesion assay of LLC cells by NETs. (K) Representative fluorescence images of the adhesion assay for DiI-labeled LLC cells trapped within PMA- or Rg1-treated neutrophils. (L) RNA-seq analysis of neutrophils. GSEA of genes related to the regulation of vascular permeability. (M) Schematic diagram of the endothelial permeability assay. (N) The effects of Rg1 or NETs on vascular permeability were tested via an endothelial permeability assay. (O-P) Representative immunofluorescence images and quantification of CD31 and FITC-dextran in the lungs ( n = 3; scale bar, 50 μm). All the data are presented as mean ± SEM. # p < 0.05, #### p < 0.0001 vs. the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. the model group.

Article Snippet: Human neutrophils were isolated from the peripheral blood of healthy subjects via a neutrophil isolation kit (P9040; Solarbio, Beijing, China).

Techniques: Permeability, CCK-8 Assay, Isolation, Transwell Invasion Assay, Immunofluorescence, Cell Adhesion Assay, Fluorescence, Labeling, RNA Sequencing, Control

Journal: Nature

Article Title: Chemically defined cytokine-free expansion of human haematopoietic stem cells.

doi: 10.1038/s41586-023-05739-9

Figure Lengend Snippet: Fig. 4 | Long-term selective expansion of functional human HSCs in 3a medium. a, The log2-transformed fold change in the expression of the indicated HSC-associated genes. Mean ± s.d. of three independent cultures. EPCR+ and EPCR− indicate CD34highEPCR+ cells and CD34highEPCR− cells, respectively. **†P = 0.0049; **‡P = 0.0043; **§P = 0.0022; ***P = 0.0006. b,c, Results from GSEA for genes differentially expressed between CD34highEPCR+ and CD34highEPCR− samples using gene sets for HSC genes (b) and mitochondrial oxidative-phosphorylation-related genes (c). Statistical significance was calculated using an empirical phenotype-based permutation test procedure38. NES, normalized enrichment score. d, Uniform manifold approximation and projection (UMAP) plot of single-cell RNA-seq data from 10-day-expanded CD34+ cord blood cells with 12 cell clusters annotated (see ‘Single-cell RNA-seq’ in Methods for details of the abbreviations used). Integrated cell map from cells cultured in PCL-PVAc-PEG-based 3a medium, StemSpan with SR-1 medium

Article Snippet: After separation, CD34+ cells were enriched using the Human CD34 Microbeads Kit (Miltenyi Biotec; 130-046-702) and MACS LS columns (Miltenyi Biotec; 130-042-401).

Techniques: Functional Assay, Transformation Assay, Expressing, Phospho-proteomics, RNA Sequencing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: Hypoxia increased the production of S100 calcium-binding protein A8 (S100A8) in neuron and microglia and induced the release of S100A8 in SH-SY5Y cells. ( A , B ) S100A8 expression (red) were detected by immunocytochemical analysis in primary cultured neurons (NeuN, neuron marker) and cultured mixed glia (Iba1, microglial marker and GFAP, astrocyte marker) exposed to hypoxic conditions for 48 h. Scheme 25 μm. S100A8 expression was detected by western blot analysis in ( C , D ) SH-SY5Y cells and ( E , F ) BV-2 cells exposed to hypoxic conditions for 48 h. ( G , H ) S100A8 protein expression in BV-2 cells were confirmed by immunocytochemistry and ( I ) S100A8 release in SH-SY5Y was measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after hypoxia. Values of * p < 0.05, ** p < 0.01, *** p < 0.001 versus control were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Cell Culture, Marker, Western Blot, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces pro-inflammatory cytokines and inflammation in BV-2 cells. BV-2 cells were stimulated with S100A8 (10 μg/mL) for 24 h. ( A ) The supernatant was collected and TNF-α and interleukin-6 (IL-6) analyzed by ELISA. ( B ) The protein and mRNA were extracted, and the expression levels of IL-1β were assessed by ELISA and RT-qPCR. ( C – E ) The protein was extracted, separated on 10% SDS-acrylamide gels (15 μg/lane) and transferred to nitrocellulose membrane. The protein expression level was detected by western blotting with anti-ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), anti-JNK and anti-p-JNK. ( F ) Cells were pre-treated with ERK inhibitor (PD98059, 20 μM), JNK inhibitor (SP600125, 10 μM) or the equivalent volume of DMSO for 1 h, then stimulated for 24 h with LPS or S100A8 for ELISA of TNF-α, IL-6. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, *** p < 0.001 versus control; ### p < 0.001 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

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

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces inflammasome priming by toll-like receptor (TLR)-4 receptors associated with ERK and JNK pathway in BV-2 cells. BV-2 cells were incubated for 24 h with LPS (1 μg/mL) or S100A8 (10 μg/mL) followed by Adenosine 5′-triphosphate disodium salt hydrate (ATP) (1 mM) for 1 h. ( A , B ) The NLRP3, ASC, and ( C , D ) cleaved caspase-1 were detected by western blotting. β-actin was used as an internal control. ( E , F ) BV-2 cells were lysed to whole lysates and IκB-α phosphorylation was analyzed by western blotting. ( G , H ) The translocation of nuclear factor- κB (NF-κB) was also detected by western blotting. BV-2 cells were lysed to cytosolic extracts and nucleic extracts. Lamin-B1 was used as internal controls. ( I , J ) BV-2 microglial cells were pre-treated with PD98059 (ERK inhibitor, 20 μM), SP600125 (JNK inhibitor, 10 μM), TAK-202 (TLR4 inhibitor, 10 μg/mL) or an equivalent volume of DMSO and stimulated for 24 h with LPS or S100A8. Cells harvested and lysed in RIPA buffer for western blotting of NLRP3. Results are from one experiment that is representative of at least three others. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Control, Phospho-proteomics, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 derived from neuronal cells induces NLRP3 inflammasome priming in microglia under hypoxic conditions. BV-2 cells were pre-treated with TAK-202 (TLR4 inhibitor, 10 μg/mL) for 1 h, then stimulated for 48 h in hypoxic condition with SH-SY5Y cells indirectly co-cultured in 0.4 μm pore transwell. ( A ) The protein expression level was detected by western blotting with NLRP3. β-actin was used as an internal control. ( B ) Quantitative analysis of NLRP3 levels. Data from three independent experiments are presented as the means ± S.D. Values of ** p < 0.01 versus control; # p < 0.05 versus co-cultured sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces apoptosis of neuronal cells in hypoxic condition. ( A , B ) SH-SY5Y cells incubated without or with S100A8 KD BV-2 cells for 48 h in hypoxic condition. Cleaved caspase-3 immunofluorescence images and were detected and quantitative analysis of the number of cleaved-caspase3-positive cells are shown in lower panel. ( C , D ) Representative Annexin-V/PI images were detected by flow cytometry. Quantitative analysis of the apoptotic rate of SH-SY5Y cells are shown in lower panel. ( E , F ) Primary neuron-glial mixed cells were transfected with S100A8 shRNA vector for 24 h followed by 48 h in hypoxic condition. Cells were harvested, and the expression protein levels of S100A8 and cleaved caspase-3 were analyzed by Western blotting. Data from three independent experiments are presented as the means ± S.D. Values of *** p < 0.001 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Transfection, shRNA, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of incubation of cell lines with rec-TF and PAR2-activation on PTEN phosphorylation. ( A ) Cells (MDA-MB-231, MCF-7, T47-D, LoVo, CaCo-2, AsPC-1 and Panc-1) were cultured in the recommended media and activated either by the addition of recombinant TF (0–1300 pg/ml) or by incubation with PAR2-agonist peptide (PAR2-AP; SLIGKV; 20 µM) and incubated for the durations shown. The cells were then lysed in electrophoresis-loading buffer and separated on a 12% (w/v) denaturing polyacrylamide gel. The proteins were then transferred to nitrocellulose membrane and blocked with TBST. The membranes were in turn probed using a rabbit anti-human phosphoSer382/Thr382/Thr383-PTEN, a polyclonal rabbit anti-human PTEN antibody, both diluted 1:2000 (v/v), or a goat anti-human GAPDH polyclonal antibody (V-18), diluted 1:4000 (v/v) in TBST. The membranes were then washed with TBST and probed with goat anti-rabbit or donkey anti-goat alkaline phosphatase-conjugated antibodies as required, diluted 1:4000 (v/v), for 90 min. Bands were then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded (micrographs are representative of 6 independent experiments; due to the number of gels the micrographs are cropped to include the main band but also to include at least three marker bands spanning the protein of interest. Full micrograph replicates are included in the ). The ratio of phospho-PTEN:Total PTEN were determined in the cell samples treated with ( B ) rec-TF (65 pg/ml) for 1 h, and ( C ) PAR2-AP for 30 min (n = 6; *p < 0.05 vs. the respective untreated samples). Similarly, the ratios of phospho-PTEN:Total PTEN were determined in the samples treated with ( D ) rec-TF (130 pg/ml) and ( E ) rec-TF (1300 pg/ml).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Incubation, Activation Assay, Phospho-proteomics, Cell Culture, Recombinant, Electrophoresis, Membrane, Western Blot, Marker

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of incubation of cell lines with rec-TF and PAR2-activation on PTEN activity and Akt inhibition. Cells (2 × 10 5 ) were treated with ( A ) rec-TF (65 pg/ml) for 1 h, or ( B ) PAR2-AP for 30 min, lysed in PhosphoSafe buffer (150 µl) and the lipid-phosphatase activity of PTEN measured using the echelon PTEN Activity ELISA kit (n = 3; *p < 0.05 vs. the respective untreated samples). Akt activity was also determined in the samples treated with ( C ) rec-TF or ( D ) PAR2-AP as above, and measured using the Akt Kinase activity assay kit according to manufacturer's instructions (n = 3; *p < 0.05 vs. the respective untreated samples). Samples of MDA-MB-231, LoVo and CaCo-2 cells were pre-incubated with a PAR2 blocking antibody (SAM-11, 20 µg/ml) or a control isotype antibody. The cells were treated with recombinant TF and ( E ) PTEN activity and ( F ) Akt activity measured as above (n = 3; *p < 0.05 vs. the respective control isotype antibody).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Incubation, Activation Assay, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Kinase Assay, Blocking Assay, Control, Recombinant

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of long-term treatment of cell lines with rec-TF on cellular PTEN antigen levels and Akt activity. Equal number of cells were lysed in PhosphoSafe buffer (150 µl) and ( A ) the level of PTEN antigen measured using a PTEN ELISA kit (n = 5; *p < 0.05 vs. the samples on first day 0). ( B ) The Akt activity was also measured using the Akt Kinase activity assay kit (n = 3; *p < 0.05 vs. the samples on first day 0). Cells (2 × 10 5 ) were cultured in the recommended media and supplemented every 2 days with rec-TF (65 pg/ml) up to 14 days. ( C ) The number of cells in the treated and untreated samples were determined using crystal violet staining (n = 3; *p < 0.05 vs. the samples on first day 0). In addition, cells were lysed in PhosphoSafe buffer and ( D ) the level of PTEN antigen and ( E ) the Akt kinase activity measured. (n = 3; *p < 0.05 vs. the samples on first day 0).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Cell Culture, Staining

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: Analysis of the proximity of PTEN and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min. The proximity between PTEN and MAGI1-3 were examined using a mouse anti-human PTEN (217702) diluted 1:100 (v/v) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (1 μg/ml) and a rabbit anti-MAGI3 antibody (1 μg/ml). The cells were then labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Ziess Axio Vert.A1 inverted fluorescence microscope with a ×40 magnification (the micrographs are representative of 10 fields of view from 4 independent experiments RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Proximity Ligation Assay, Activation Assay, Incubation, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: Analysis of the interaction of PTEN and MAGI1-3 and the influence of PAR2 activation. MDA-MB-231 (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and analysed by PLA as described in Fig. . The number of red fluorescent events and nuclei were determined using ImageJ, in 10 fields of view from each assay for ( A ) the interactions of PTEN with MAGI1-3 in non-activated and at 20 min post-activation. ( B ) In addition, the interaction of PTEN and MAGI1 and 2, was measured at intervals up to 30 min (n = 3; *p < 0.05 vs. the non-activated sample). MAGI2 was immunoprecipitated from cell lysates with an anti-MAGI2 (C3; 4 µg) antibody using protein A-magnetic beads. The MDA-MB-231 lysate samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and ( C ) examined for PTEN and MAGI2 by western blot using a mouse anti-PTEN antibody (217702) and a rabbit anti-MAGI2 antibody. ( D ) The ratio of the PTEN band densities were normalised against those of MAGI2 in the same co-immunprecipitated samples (n = 3; *p < 0.05 vs. the non-activated sample). Similarly, the LoVo lysate samples were used to immunoprecipitate MAGI2 and ( E ) examined for PTEN and MAGI2 by western blot using a mouse anti-PTEN antibody (217702) and a rabbit anti-MAGI2 antibody. ( F ) The ratio of the PTEN band densities were normalised against those of MAGI2 in the same co-immunprecipitated samples (n = 3; *p < 0.05 vs. the non-activated sample). Full micrograph replicates are included in the s .

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot