Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments

doi: 10.1111/j.1538-7836.2011.04438.x

Figure Lengend Snippet: Inhibitory effect of β15-42-containing fibrin fragments on NDSK-II dependent transendothelial migration of neutrophils in vitro. HUVECs were grown to confluency on gelatin-coated cell culture inserts. Calcein AM-labeled HL-60 cells (panel A) were differentiated into neutrophil-like cells (panel B) and added to the upper chambers (inserts) on top of the HUVEC monolayer in the presence of 1.5 μM NDSK-II without or with 10 μM β15-42, (β15-44)2, or (β15-66)2 fragments. The cells were allowed to migrate into the lower chambers for 4 h at 37 °C, collected, and measured by fluorescence at 530 nm (panel C). The results in panel C are expressed as % of NDSK-II dependent cell migration that was calculated by subtracting migrated cells in the absence of NDSK-II from total migrated cells. The graph shows combined data obtained in 4 independent experiments; bars denote means ± the standard error (n = 15-20 wells for each condition); ***, P < 0.001. The inset in panel C shows dose-dependence of the inhibitory effect of the β15-42 and (β15-44)2 fragments (empty and filled circles, respectively) on NDSK-II dependent neutrophil transmigration.

Article Snippet: Differentiated HL-60 cells were labeled with Calcein AM fluorescent dye (BD Biosciences) in serum-free IMDM medium, as recommended by the manufacturer.

Techniques: Migration, In Vitro, Cell Culture, Labeling, Fluorescence, Transmigration Assay

Journal: Cells

Article Title: Evidence for a Role of the Long Non-Coding RNA ITGB2-AS1 in Eosinophil Differentiation and Functions

doi: 10.3390/cells13231936

Figure Lengend Snippet: The effect of ITGB2-AS1 lncRNA deficiency on eosinophil differentiation, granulogenesis, and EPX expression. ( A – F ) HL-60c15 cells were differentiated into ELCs in the presence of sodium butyrate and IL-5 for up to 6 days. ( A ) Cell morphology. ( Left ) Representative images of differentiating HL-60c15 following Hemacolor Rapid staining at the indicated days of differentiation. Images were acquired with the automatic digital slide scanner Pannoramic MIDI II. Intracellular granules are indicated by black arrows. Scale bars, 10 µm. ( Right ) The frequency of granulated cells was evaluated manually by light microscopy using a C plan 100×/1.25 Oil objective ( n = 4). ( B , C ) Flow cytometry. The frequency of HL-60c15 cell differentiation was assessed by CD11b ( B ) and CCR3 ( C ) surface expression after exclusion of dead cells ( n = 4). ( D ) Quantitative PCR. Relative RNA levels of the granule protein EPX in differentiating HL-60c15 cells after the indicated days of differentiation. EPX RNA levels were normalized using the geometric mean of the reference genes GAPDH , UBC , and HPRT1 and presented relative to shControl cells at day 0 of differentiation ( n ≥ 3). ( E ) Immunoblotting. Protein lysates were obtained from differentiating HL-60c15 cells at the indicated days of differentiation. EPX was detected using a monoclonal mouse anti-EPX antibody. GAPDH protein levels served as loading controls. Lysates from human blood eosinophils (Human Eos) were used as a positive control for the presence of EPX. A representative immunoblot of three independent experiments is shown. ( F ) Confocal microscopy. Differentiating HL-60c15 cells were stained for the eosinophil granule protein EPX and the nuclei using monoclonal mouse anti-EPX antibody and Hoechst 33342, respectively. ( Left ) Representative images of the presence of EPX in HL-60c15 cells at the indicated days of differentiation. ( Right ) Quantification of the mean fluorescence intensity (MFI) of intracellular EPX. Cells were delimited using “Surfaces” mode in Imaris, followed by EPX (green channel) MFI quantification ( n = 4, with ≥42 cells per condition). Scale bars, 10 µm. Values are means ± SEM. ns, not significant; * p < 0.05. **** p < 0.0001. Significances in black illustrate the significance of shControl cells compared with undifferentiated (day 0) shControl cells. Significances in green denote the significance of shITGB2-AS1 cells compared with shITGB2-AS1 cells at day 0. Significances in red illustrate the significant difference between the shControl and shITGB2-AS1 cells. Abbreviations: ELC, eosinophil-like cell; EPX, eosinophil peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT1, hypoxanthine-guanine phosphoribosyltransferase 1; lncRNA, long non-coding RNA; kDa, kilodalton; MFI, mean fluorescence intensity; RNA, ribonucleic acid; UBC, ubiquitin C.

Article Snippet: Considering the challenges associated with the genetic modification of human eosinophils and the inability to conduct functional screenings with mouse eosinophils due to the specificity of the lncRNA ITGB2-AS1 to human cells, we employed the human promyelocytic leukemia cell line HL-60 clone 15 (HL-60c15, ATCC CRL-1964) for genetic modifications and subsequent functional studies [ , ].

Techniques: Expressing, Staining, Light Microscopy, Flow Cytometry, Cell Differentiation, Real-time Polymerase Chain Reaction, Western Blot, Positive Control, Confocal Microscopy, Fluorescence, Ubiquitin Proteomics

Journal: Cells

Article Title: Evidence for a Role of the Long Non-Coding RNA ITGB2-AS1 in Eosinophil Differentiation and Functions

doi: 10.3390/cells13231936

Figure Lengend Snippet: Expression of eosinophil-related proteins shown to be co-expressed with the lncRNA ITGB2-AS1 in the correlation network analysis. ( A – E ) Flow cytometry. HL-60c15 cells were differentiated into ELCs in the presence of sodium butyrate and IL-5 for up to 6 days. The surface protein expression of ITGB2 ( A ), CCR1 ( B ), CD48 ( C ), CD52 ( D ), and CXCR3 ( E ) was assessed after the exclusion of dead cells ( n ≥ 3). ( A – E ) ( Left ) Frequency of live cells expressing the proteins at the plasma membrane. ( Right ) Surface protein expression levels are represented as MFI in live cells. Values are means ± SEM. ns, not significant; * p < 0.05. ** p < 0.01; *** p < 0.001; **** p < 0.0001. Significances in black illustrate the significance of shControl cells compared with undifferentiated (day 0) shControl cells. Significances in green denote the significance of shITGB2-AS1 cells compared with undifferentiated shITGB2-AS1 cells. Significances in red illustrate a significant difference between the shControl and shITGB2-AS1 cells. Abbreviations: CXCR3, C-X-C motif chemokine receptor 3; ELC, eosinophil-like cell; ITGB2, integrin subunit beta 2; MFI, mean fluorescence intensity.

Article Snippet: Considering the challenges associated with the genetic modification of human eosinophils and the inability to conduct functional screenings with mouse eosinophils due to the specificity of the lncRNA ITGB2-AS1 to human cells, we employed the human promyelocytic leukemia cell line HL-60 clone 15 (HL-60c15, ATCC CRL-1964) for genetic modifications and subsequent functional studies [ , ].

Techniques: Expressing, Flow Cytometry, Clinical Proteomics, Membrane, Fluorescence

Journal: Cells

Article Title: Evidence for a Role of the Long Non-Coding RNA ITGB2-AS1 in Eosinophil Differentiation and Functions

doi: 10.3390/cells13231936

Figure Lengend Snippet: The impact of ITGB2-AS1 lncRNA deficiency on eosinophil degranulation and ROS production. ( A – C ) HL-60c15 cells differentiated for 6 days in the presence of sodium butyrate and IL-5 were primed with GM-CSF or IL-3 for 20 min and subsequently stimulated with C5a for 30 min at 37 °C. ( A , B ) Degranulation assays. ( A ) Flow cytometry. Following the aforementioned stimulation, eosinophil degranulation was assessed by measuring CD63 surface expression ( n = 5). ( Right ) A representative histogram of flow cytometry data is shown for each condition. ( B ) EPX assay. Subsequent to the previously mentioned stimulation, the release of the eosinophil granule protein EPX into the supernatant was evaluated by determining EPX activity using the peroxidase substrate O-phenylenediamine (OPD) and measuring absorbance at 492 nm ( n = 5). ( C ) ROS production. Following the above-mentioned stimulation, ROS production was assessed by measuring DHR123 fluorescence with a spectrofluorometer ( n = 8). Values are means ± SEM. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Significances in black illustrate the significance of shControl cells compared with untreated shControl cells. Significances in green denote the significance of shITGB2-AS1 cells compared with untreated shITGB2-AS1 cells. Significances in red illustrate the significant difference between the shControl and shITGB2-AS1 cells for the same condition. Abbreviations: abs, absorbance; C5a, complement component 5a; EPX, eosinophil peroxidase; GM-CSF, granulocyte-macrophage colony-stimulating factor; DHR123, dihydrorhodamine 123; IL-3, interleukin 3; IL-5, interleukin 5; ITGB2-AS1, ITGB2 antisense RNA 1; MFI, mean fluorescence intensity; OPD, O-phenylenediamine; RFU, relative fluorescence units; ROS, reactive oxygen species.

Article Snippet: Considering the challenges associated with the genetic modification of human eosinophils and the inability to conduct functional screenings with mouse eosinophils due to the specificity of the lncRNA ITGB2-AS1 to human cells, we employed the human promyelocytic leukemia cell line HL-60 clone 15 (HL-60c15, ATCC CRL-1964) for genetic modifications and subsequent functional studies [ , ].

Techniques: Flow Cytometry, Expressing, Activity Assay, Fluorescence

Journal:

Article Title: Aspergillus fumigatus AcuM Regulates both Iron Acquisition and Gluconeogenesis

doi: 10.1111/j.1365-2958.2010.07389.x

Figure Lengend Snippet: Cluster analysis of SreA-responsive gene expression in the ΔacuM mutant. Microarray data comparing the response of the ΔacuM mutant with Af293 (WT) and the ΔacuM∷acuM complemented strain (Comp) after 18 and 24 h incubation in RPMI 1640 medium at 37°C are shown. The bar at the top indicates the colors that correspond to the observed expression ratios. The genes are displayed in the order of their chromosomal location and the vertical colored boxes indicate gene clusters. The numbers in these boxes correspond to the SreA-responsive gene clusters in (Schrettl et al., 2008). The gene names in blue font denote genes that had significantly reduced transcript levels in the ΔacuM mutant compared to the control strains.

Article Snippet: Next, 10 3 swollen conidia were added to polypropylene tubes containing differentiated HL-60 cells in 1 ml RPMI 1640 medium supplemented with 5% pooled human serum (Sigma-Aldrich) to achieve a target to effector cell ratio of 1:50.

Techniques: Expressing, Mutagenesis, Microarray, Incubation