Journal: Journal of Virology

Article Title: Foot-and-Mouth Disease Virus Exhibits an Altered Tropism in the Presence of Specific Immunoglobulins, Enabling Productive Infection and Killing of Dendritic Cells ▿

doi: 10.1128/JVI.02180-10

Figure Lengend Snippet: FMDV infection of moDC is closely associated with cell death and reduced ability to stimulate FMDV-specific T cell proliferation. (A) MoDC were exposed to medium, O1K-Cad2, or O1K-Cad2 IC at an MOI of 10 for 6 h. Phase-contrast microscopy captured the high levels of dying cells, including some exhibiting classical apoptotic morphology (high power; right). (B) MoDC were exposed to medium, O1K-Cad2, or O1K-Cad2 IC at an MOI of 10 for 1 to 8 h. Samples of each culture were labeled with antibodies against FMDV NS protein 3A or with annexin V conjugated to APC, and PI. Death was defined as staining with annexin V, PI, or both. The data presented are relative to medium-only values; R = 0.929 (Pearson's correlation coefficient) for the linear relationship between the proportion of cells positive for FMDV NS proteins and the proportion dying. (C) Purified CD4+ T cells from an FMDV-vaccinated animal were added to autologous moDC at a ratio of 1 DC to 100 T cells, with either medium, a sample of the antigen used for vaccination, purified O1K-Cad2, or purified O1K-Cad2 in IC at an MOI of 5. After 5 days, [3H]thymidine was added to each well and incubated overnight before harvesting and liquid scintillation counting of the cells for incorporated radioactivity. Pokeweed mitogen (PWM) was used as a positive control, and in all cases, counts exceeded 350,000 cpm (not shown). A representative data set is presented as cpm ± SD. *, P = 0.02; Student's t test. All 3 replicates using cells from 2 different animals were significant at this level or above.

Article Snippet: To detect dying cells in moDC cultures, 5 × 10 5 cells were pelleted by centrifugation and resuspended in annexin V binding buffer (MBL, Nagoya, Japan).

Techniques: Infection, Microscopy, Labeling, Staining, Purification, Incubation, Radioactivity, Positive Control

Journal: Scientific Reports

Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

doi: 10.1038/s41598-017-08392-1

Figure Lengend Snippet: Effects of sustained ciprofloxacin exposure on Jurkat cells. (a,b) Viability of Jurkat cells with/without exposure to ciprofloxacin (10 µg/mL for >14 days) was analyzed by flow cytometry after staining with annexinV-FITC and propidium iodide (PI). (a) Mean values+/− S.D. (error bars) of two independent experiments are shown in the histogram plot. AxV: annexinV. (b) Representative dot plots showing the four quadrants of annexinV-FITC and PI stained Jurkat cells. (c) Exosomes (EXOs) derived from ciprofloxacin-exposed/unexposed Jurkat cells were conjugated onto latex beads and characterized by flow cytometry after staining with annexinV-FITC, anti-CD63-PE, PI or anti-histone H2B-FITC. The background fluorescence of stained latex beads is indicated by grey histograms. (d) Microvesicles (MVs) and apoptotic bodies (APOs) were labeled directly with annexinV-FITC and PI. The background fluorescence of EVs is indicated by grey histograms.

Article Snippet: AnnexinV-labeled cells were diluted 6x with annexinV binding buffer (BD Biosciences, San Jose, CA) to the final volume of 300 µL, and PI was added to the samples in a final concentration of 1 µg/mL before flow cytometry measurements.

Techniques: Flow Cytometry, Staining, Derivative Assay, Fluorescence, Labeling

Journal: Scientific Reports

Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

doi: 10.1038/s41598-017-08392-1

Figure Lengend Snippet: Analysis of ciprofloxacin-induced release of DNA associated with extracellular vesicles (EVs). (a) DNA content of size-based EV fractions determined with/without DNase I digestion of EVs. Concentration values represent the amount of EV-associated DNA (eluted in 3 0 µL) isolated from the conditioned medium of 2.5 × 10 7 Jurkat cells. Plotted values are presented as the mean+/− S.D. (error bars) of 8 independent experiments. (**P < 0.01, Friedman test, One-way ANOVA). (b) DNA amounts before and after the digestion of EVs by DNase I. The paired measurements (n = 8) for APOs, MVs and EXOs are indicated by lines (**P < 0.01, Wilcoxon signed rank test). APO: apoptotic body, MV: microvesicle, EXO: exosome (c) The presence of EXOs both in undigested and DNase I digested samples was confirmed by flow cytometry. Latex-bound EXOs were stained with annexinV-FITC and an anti-CD63-PE antibody. Dot plots are representative of three independent experiments. (d) Optiprep TM density gradient fractions of the 100,000 g pellet (containing EXOs) were re-pelleted, conjugated onto latex beads and stained with an anti-CD63-PE antibody for flow cytometry. (e) The same latex-bound Optiprep TM density gradient fractions were also stained by propidium iodide (PI). The percentages of PI positive events are shown above a threshold (horizontal line, determined by measuring labeled latex beads without conjugated density gradient fractions).

Article Snippet: AnnexinV-labeled cells were diluted 6x with annexinV binding buffer (BD Biosciences, San Jose, CA) to the final volume of 300 µL, and PI was added to the samples in a final concentration of 1 µg/mL before flow cytometry measurements.

Techniques: Concentration Assay, Isolation, Flow Cytometry, Staining, Labeling

Journal: Scientific Reports

Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

doi: 10.1038/s41598-017-08392-1

Figure Lengend Snippet: Association of DNA with EXO surface is abolished by high salt concentration. Ciprofloxacin-exposed Jurkat cell exosomes (EXOs) were conjugated onto latex beads and washed using annexinV binding buffer supplemented with 0.1 M, 1 M or 2 M NaCl. All latex-bound EXO samples were re-suspended in annexinV binding buffer, and labeled with propidium iodide (PI), annexinV-FITC and an anti-CD63-PE antibody for flow cytometry. (a) Differences of geometric mean fluorescence values between EXOs washed in high salt concentration buffers and controls are shown. Plotted values are presented as the mean+/− S.D. (error bars) of three independent experiments. *P < 0.05, Friedman test, One-way ANOVA (b) Density plots show PI, annexinV-FITC and anti-CD63-PE fluorescence of latex-bound EXOs as a function of SSC parameter. Horizontal lines indicate fluorescence threshold of labeled EXO-free latex beads.

Article Snippet: AnnexinV-labeled cells were diluted 6x with annexinV binding buffer (BD Biosciences, San Jose, CA) to the final volume of 300 µL, and PI was added to the samples in a final concentration of 1 µg/mL before flow cytometry measurements.

Techniques: Concentration Assay, Binding Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

doi: 10.1038/s41598-017-08392-1

Figure Lengend Snippet: Flow cytometry analysis of extracellular vesicles (EVs) derived from ciprofloxacin-exposed control, activated or apoptotic Jurkat cells. EVs were stained by annexinV-FITC and propidium iodide (PI) for flow cytometry and analyzed before and after detergent lysis with 0.1% Triton X-100. Apoptotic bodies (APOs) and microvesicles (MVs) were labeled and measured directly, whereas exosomes (EXOs) were conjugated onto latex beads before staining. Density plots show annexinV-FITC and PI positivity of EVs derived from ciprofloxacin-exposed control, activated and apoptotic Jurkat cells. The percentages of positive events (above the threshold represented by a black line) are shown in the plots.

Article Snippet: AnnexinV-labeled cells were diluted 6x with annexinV binding buffer (BD Biosciences, San Jose, CA) to the final volume of 300 µL, and PI was added to the samples in a final concentration of 1 µg/mL before flow cytometry measurements.

Techniques: Flow Cytometry, Derivative Assay, Staining, Lysis, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: TRIP6 regulates the proliferation, migration, invasion and apoptosis of osteosarcoma cells by activating the NF-κB signaling pathway

doi: 10.3892/etm.2020.8466

Figure Lengend Snippet: TRIP6 plays a role in Os cell proliferation. (A) Western blot analysis of TRIP6 protein expression level in TRIP6 silenced-U2OS cell lines and (B) quantification. (C) Western blot analysis of TRIP6 protein expression level in TRIP6 transduced-MG63 cell lines and (D) quantification. Data were normalized to GAPDH and are presented as the mean ± SD from three independent experiments. MTT assay results from (E) U2OS cells and (F) MG63 cells indicated that growth rates increased in TRIP6-overexpressing cells and decreased in TRIP6-silenced cells. (G) U2OS cells colony formation assay (H) results and (I) MG63 cells colony formation assay (J) results indicated that growth rates increased in TRIP6-overexpressing cells and decreased in TRIP6-silenced cells. Colony formation, classified as colonies >0.1 mm diameter, was quantified after 12 days of culture. Apoptosis of U2-OS and MG63 cells analyzed by flow cytometry after different treatments. (K) The apoptotic rate was decreased in U2OS-NC cells (6.42%) (L) compared with U2OS-TRIP6-siRNA cells (15.61%). (M) The apoptosis rates of (K) and (L) were statistically significant. (N) The apoptotic rate was 7.16% in the MG63-Vector group and (O) 3.48% in the MG63-TRIP6-flag group. (P) The apoptosis rates of (N) and (O) were statistically significant. *P<0.05 vs. control group. Os, osteosarcoma; TRIP6, thyroid hormone receptor-interacting protein 6; NC, negative control; siRNA, small interfering RNA.

Article Snippet: After cells were harvested, washed twice with PBS and resuspended in 450 μl binding buffer (Annexin V-FITC cell apoptosis assay kit; Beyotime Institute of Biotechnology), Annexin V-FITC was added (5 μl/well).

Techniques: Western Blot, Expressing, MTT Assay, Colony Assay, Flow Cytometry, Plasmid Preparation, Control, Negative Control, Small Interfering RNA