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  • 99
    Thermo Fisher propidium iodi de
    Chromosomal abnormalities. Metaphase chromosomal spreads were prepared from MSCs with the following genotypes a pure wild-type C57/Bl6 (C57 WT) cells; c EWS-FLI1 p53−/− cells expressing random control shRNA (Ctrl shRNA cells); and e EWS-FLI1 p53−/− cells expressing Stag2 shRNA (Stag2 shRNA cells). Examination of 125 metaphase spreads showed more abnormal metaphases for Ctrl shRNA and Stag2 shRNA cells compared to C57 WT cells. Ctrl shRNA and Stag2 shRNA cells exhibited frequent non-reciprocal translocations (red arrows), chromosomal fragments (blue arrows) and chromosomal breaks (green arrows). However, there was no significant difference between Ctrl shRNA and Stag2 shRNA cells in terms of percentage of aberrant metaphases (34% vs. 34%, respectively), chromosomal breaks (18% vs. 16%, respectively), and chromosomal fusions/translocations (24% vs. 24%). b The cell cycle distribution of C57/Bl6 WT cells stained with <t>propidium</t> iodide (PI) showed 89.1% of cells in G0-G1, 2.1% in S, and 7.6% in G2-M phases. Cell cycle distribution of Ctrl shRNA cells d and Stag2 shRNA cells f showed a higher fraction of non-G0-G1 cells compared to the control C57 WT cells. The cell cycle distribution of Ctrl shRNA cells was not statistically different compared to Stag2 shRNA cells
    Propidium Iodi De, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore propidium iodine
    Penetration of CIGB-210 into the MT4 cell line. ( A , B ) CIGB-210 uptake assessed by flow cytometry. MT4 cells were incubated with 10, 20 or 40 µM of carboxyfluorescein labeled CIGB-210 for 15 min, 60 min or 24 h. External fluorescence was quenched by addition of 0.4% Trypan blue. CC: Control untreated MT4 cells; PP: carboxyfluorescein labeled peptide containing the Tat cell penetrating peptide used as positive control; ( A ) Flow cytometry histograms; and ( B ) percentage of fluorescent cells for each experimental condition. Data represent the mean ± standard deviation of three experiments performed in triplicate; ( C – E ) CIGB-210 uptake assessed by fluorescence microscopy. MT4 cells were treated with: 10 µM biotinylated CIGB-210 ( E ); 10 µM biotinylated Tat cell penetrating peptide ( D ); or medium ( C ) for 24 h, fixed and visualized with a streptavidin-FITC conjugate. The nucleus was stained with <t>propidium</t> iodine ( red ). 100× magnification.
    Propidium Iodine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore propridium iodide
    Penetration of CIGB-210 into the MT4 cell line. ( A , B ) CIGB-210 uptake assessed by flow cytometry. MT4 cells were incubated with 10, 20 or 40 µM of carboxyfluorescein labeled CIGB-210 for 15 min, 60 min or 24 h. External fluorescence was quenched by addition of 0.4% Trypan blue. CC: Control untreated MT4 cells; PP: carboxyfluorescein labeled peptide containing the Tat cell penetrating peptide used as positive control; ( A ) Flow cytometry histograms; and ( B ) percentage of fluorescent cells for each experimental condition. Data represent the mean ± standard deviation of three experiments performed in triplicate; ( C – E ) CIGB-210 uptake assessed by fluorescence microscopy. MT4 cells were treated with: 10 µM biotinylated CIGB-210 ( E ); 10 µM biotinylated Tat cell penetrating peptide ( D ); or medium ( C ) for 24 h, fixed and visualized with a streptavidin-FITC conjugate. The nucleus was stained with <t>propidium</t> iodine ( red ). 100× magnification.
    Propridium Iodide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam propidium iodide
    Fluorescence micrograph of <t>propidium</t> iodide (PI) cellular uptake in MCF-7 cells by electroporation using 50 pulses and frequency 1 Hz in the absence ( a ) and presence ( b ) of multi-walled carbon nanotubes (MWCNTs) (30 μg/mL). The field intensity of main pulses (E M ) was 20 V/cm and the alignment field (E A ) intensity was 15 V/cm. Cell suspension samples in the same density (10 6 cells/mL) were treated as described above and stained with PI and viewed under microscope as described in the Experimental Section . As cell density was identical in both samples, only fluorescence images were shown here.
    Propidium Iodide, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam propidium iodide abcam
    Annexin-V/Alexa Fluor™ 647 and <t>Propidium</t> iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.
    Propidium Iodide Abcam, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision propidium iodide
    SU11652 induces apoptosis of MV - 4 - 11 cells. MV-4-11 cells were incubated with 0, 10 and 100 nM SU11652 for 24 hours. Cells were stained with Cy5-labeled annexin V and <t>propidium</t> iodide, followed by analyses with a flow cytometry. Percentages of annexin V-positive and propidium iodide-negative cells, that is, apoptotic cells, are indicated.
    Propidium Iodide, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Chromosomal abnormalities. Metaphase chromosomal spreads were prepared from MSCs with the following genotypes a pure wild-type C57/Bl6 (C57 WT) cells; c EWS-FLI1 p53−/− cells expressing random control shRNA (Ctrl shRNA cells); and e EWS-FLI1 p53−/− cells expressing Stag2 shRNA (Stag2 shRNA cells). Examination of 125 metaphase spreads showed more abnormal metaphases for Ctrl shRNA and Stag2 shRNA cells compared to C57 WT cells. Ctrl shRNA and Stag2 shRNA cells exhibited frequent non-reciprocal translocations (red arrows), chromosomal fragments (blue arrows) and chromosomal breaks (green arrows). However, there was no significant difference between Ctrl shRNA and Stag2 shRNA cells in terms of percentage of aberrant metaphases (34% vs. 34%, respectively), chromosomal breaks (18% vs. 16%, respectively), and chromosomal fusions/translocations (24% vs. 24%). b The cell cycle distribution of C57/Bl6 WT cells stained with propidium iodide (PI) showed 89.1% of cells in G0-G1, 2.1% in S, and 7.6% in G2-M phases. Cell cycle distribution of Ctrl shRNA cells d and Stag2 shRNA cells f showed a higher fraction of non-G0-G1 cells compared to the control C57 WT cells. The cell cycle distribution of Ctrl shRNA cells was not statistically different compared to Stag2 shRNA cells

    Journal: BMC Cancer

    Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

    doi: 10.1186/s12885-019-6465-8

    Figure Lengend Snippet: Chromosomal abnormalities. Metaphase chromosomal spreads were prepared from MSCs with the following genotypes a pure wild-type C57/Bl6 (C57 WT) cells; c EWS-FLI1 p53−/− cells expressing random control shRNA (Ctrl shRNA cells); and e EWS-FLI1 p53−/− cells expressing Stag2 shRNA (Stag2 shRNA cells). Examination of 125 metaphase spreads showed more abnormal metaphases for Ctrl shRNA and Stag2 shRNA cells compared to C57 WT cells. Ctrl shRNA and Stag2 shRNA cells exhibited frequent non-reciprocal translocations (red arrows), chromosomal fragments (blue arrows) and chromosomal breaks (green arrows). However, there was no significant difference between Ctrl shRNA and Stag2 shRNA cells in terms of percentage of aberrant metaphases (34% vs. 34%, respectively), chromosomal breaks (18% vs. 16%, respectively), and chromosomal fusions/translocations (24% vs. 24%). b The cell cycle distribution of C57/Bl6 WT cells stained with propidium iodide (PI) showed 89.1% of cells in G0-G1, 2.1% in S, and 7.6% in G2-M phases. Cell cycle distribution of Ctrl shRNA cells d and Stag2 shRNA cells f showed a higher fraction of non-G0-G1 cells compared to the control C57 WT cells. The cell cycle distribution of Ctrl shRNA cells was not statistically different compared to Stag2 shRNA cells

    Article Snippet: Cells were centrifuged and resuspended in 1 mL PBS mixed with 50 μg/ml propidium iodide (Invitrogen, Carlsbad, California, USA).

    Techniques: Expressing, shRNA, Staining

    Effect of P-cresol on HUVEC cell cycle. HUVEC were incubated in serum-free RPMI 1640 for 24 h and treated with unbound (A) and protein-bound (B) P-cresol (20-80 μg/ml, in RPMI 1640 2% FBS) for 72 h. Cells were stained with propidium iodide and the cell cycle was analyzed by flow cytometry for DNA content. Representative cytometric profiles and percentages of each phase were shown (A and B). The program Cell Quest was used for acquisition and analysis of the FACS scans. Data were expressed as means ± SD. G 0 /G 1 = cells in G 0 /G 1 cell cycle phases, S = cells in S cell cycle phase, G 2 /M = cells in G 2 /M cell cycle phases.

    Journal: American Journal of Translational Research

    Article Title: Protein-bound P-cresol inhibits human umbilical vein endothelial cell proliferation by inducing cell cycle arrest at G0/G1

    doi:

    Figure Lengend Snippet: Effect of P-cresol on HUVEC cell cycle. HUVEC were incubated in serum-free RPMI 1640 for 24 h and treated with unbound (A) and protein-bound (B) P-cresol (20-80 μg/ml, in RPMI 1640 2% FBS) for 72 h. Cells were stained with propidium iodide and the cell cycle was analyzed by flow cytometry for DNA content. Representative cytometric profiles and percentages of each phase were shown (A and B). The program Cell Quest was used for acquisition and analysis of the FACS scans. Data were expressed as means ± SD. G 0 /G 1 = cells in G 0 /G 1 cell cycle phases, S = cells in S cell cycle phase, G 2 /M = cells in G 2 /M cell cycle phases.

    Article Snippet: Propidium iodide (PI) was obtained from Invitrogen (Carlsbad, California, USA).

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry, FACS

    Autophagy disruption delays atrophy and enhances cell death in both the presence and absence of growth factor A. Bcl-2-expressing cells were cultured for four days in the presence or absence of 4OHT to delete Atg3, then cultured in the presence or absence of both 4OHT and IL3. Cell size of Bcl-2-expressing cells was assessed by forward scan flow cytometry. B. Control NGF or Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT, then cultured in the presence or absence of both 4OHT and IL3, and survival of control NGF and Bcl-2-expressing cells was assessed by propidium iodide exclusion flow cytometry. Note different time scales . C . Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT to prevent autophagy, and withdrawn from IL3 for indicated times. Mcl-1, Puma, Bim, and Bax, were analyzed by immunoblot. Means and standard deviations of triplicate samples are shown. Data shown are representative of three or more experiments. Asterisks denote p

    Journal: Oncogene

    Article Title: Autophagy is Essential to Suppress Cell Stress and to Allow BCR-Abl-Mediated Leukemogenesis

    doi: 10.1038/onc.2010.561

    Figure Lengend Snippet: Autophagy disruption delays atrophy and enhances cell death in both the presence and absence of growth factor A. Bcl-2-expressing cells were cultured for four days in the presence or absence of 4OHT to delete Atg3, then cultured in the presence or absence of both 4OHT and IL3. Cell size of Bcl-2-expressing cells was assessed by forward scan flow cytometry. B. Control NGF or Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT, then cultured in the presence or absence of both 4OHT and IL3, and survival of control NGF and Bcl-2-expressing cells was assessed by propidium iodide exclusion flow cytometry. Note different time scales . C . Bcl-2-expressing A3C cells were cultured for two days with ethanol or 4OHT to prevent autophagy, and withdrawn from IL3 for indicated times. Mcl-1, Puma, Bim, and Bax, were analyzed by immunoblot. Means and standard deviations of triplicate samples are shown. Data shown are representative of three or more experiments. Asterisks denote p

    Article Snippet: Cell death was by flow cytometry analysis (FACScan, Becton Dickinson) using propidium iodide exclusion (PI, 1 μg / ml, Invitrogen) and analysis with Flowjo software (Treestar Inc., Ashland, OR).

    Techniques: Expressing, Cell Culture, Flow Cytometry, Cytometry

    T-cell development is arrested at the DN1-to-DN2 transition in Id1 transgenic mice. (A) Total thymocytes from wild-type (WT), Id1 tg and Id1 tg/tg mice in the FVB/N background were analyzed for expression of CD19 (top) and NK1.1 (bottom). Numbers show the percentages of CD19 + or NK1.1 + cells within propidium iodide-negative live-cell gates. Average total thymocyte counts ± standard deviations are shown beneath the genotypes of the mice. (B) CD4 and CD8 expression on CD19 − thymocytes from mice as described for panel A. Numbers show percentages of cells in each quadrant. Levels of CD44 and CD25 expression on thymocytes negative for B220, CD4, CD8, TCRγδ, DX5, NK1.1, and Mac1 staining (Lin − ) are shown at the bottom. The DN1′ cell gate is shown as a circle, and percentage of cells within the gate are indicated. (C) CD44 and CD25 expression on thymocytes of mice in the C57BL/6 background. Average total thymocyte counts ± standard deviations are shown beneath the genotypes of the mice.

    Journal: Molecular and Cellular Biology

    Article Title: Id1 Attenuates Notch Signaling and Impairs T-Cell Commitment by Elevating Deltex1 Expression

    doi: 10.1128/MCB.00119-09

    Figure Lengend Snippet: T-cell development is arrested at the DN1-to-DN2 transition in Id1 transgenic mice. (A) Total thymocytes from wild-type (WT), Id1 tg and Id1 tg/tg mice in the FVB/N background were analyzed for expression of CD19 (top) and NK1.1 (bottom). Numbers show the percentages of CD19 + or NK1.1 + cells within propidium iodide-negative live-cell gates. Average total thymocyte counts ± standard deviations are shown beneath the genotypes of the mice. (B) CD4 and CD8 expression on CD19 − thymocytes from mice as described for panel A. Numbers show percentages of cells in each quadrant. Levels of CD44 and CD25 expression on thymocytes negative for B220, CD4, CD8, TCRγδ, DX5, NK1.1, and Mac1 staining (Lin − ) are shown at the bottom. The DN1′ cell gate is shown as a circle, and percentage of cells within the gate are indicated. (C) CD44 and CD25 expression on thymocytes of mice in the C57BL/6 background. Average total thymocyte counts ± standard deviations are shown beneath the genotypes of the mice.

    Article Snippet: After staining with appropriate antibodies, dead cells were excluded based on propidium iodide incorporation (Molecular Probes, Eugene, OR).

    Techniques: Transgenic Assay, Mouse Assay, Expressing, Staining

    Protein expression and functional detection of FFAR1/GPR40 in BUVEC monolayers. a Western blot of bovine FFAR1/GPR40. Total protein extract from LoVo cells was used as the positive control for two BUVEC isolates. 50 μg of total protein was loaded onto a 6 % polyacrylamide gel. b FFAR1/GPR40 immunofluorescence. Left image , nuclear signals of cells incubated with propidium iodide (PI); inset image: only the secondary antibody was used, as the negative control, bar = 20 μm. Middle image , FFAR1/GPR40 detected with rabbit anti-GPR40 antiserum and an anti-rabbit IgG secondary antibody. Right image , nuclear signals and GPR40 images are superimposed, bar = 20 nm. c Representative time course of the Fura-2/AM ratio in BUVECs exposed to 1 % DMSO or 100 μM TAK-875 at the time indicated by the arrow. d Cells were exposed to DMSO or TAK-875. The area under the curve (AUC) was calculated during the first 100 s after exposure to the stimulus. The data are expressed as the means ± SEM of at least three independent experiments. ** p

    Journal: BMC Veterinary Research

    Article Title: Differential intracellular calcium influx, nitric oxide production, ICAM-1 and IL8 expression in primary bovine endothelial cells exposed to nonesterified fatty acids

    doi: 10.1186/s12917-016-0654-3

    Figure Lengend Snippet: Protein expression and functional detection of FFAR1/GPR40 in BUVEC monolayers. a Western blot of bovine FFAR1/GPR40. Total protein extract from LoVo cells was used as the positive control for two BUVEC isolates. 50 μg of total protein was loaded onto a 6 % polyacrylamide gel. b FFAR1/GPR40 immunofluorescence. Left image , nuclear signals of cells incubated with propidium iodide (PI); inset image: only the secondary antibody was used, as the negative control, bar = 20 μm. Middle image , FFAR1/GPR40 detected with rabbit anti-GPR40 antiserum and an anti-rabbit IgG secondary antibody. Right image , nuclear signals and GPR40 images are superimposed, bar = 20 nm. c Representative time course of the Fura-2/AM ratio in BUVECs exposed to 1 % DMSO or 100 μM TAK-875 at the time indicated by the arrow. d Cells were exposed to DMSO or TAK-875. The area under the curve (AUC) was calculated during the first 100 s after exposure to the stimulus. The data are expressed as the means ± SEM of at least three independent experiments. ** p

    Article Snippet: The propidium iodide signals were detected with a fluorescence multiplate reader (Varioskan®, Thermo Scientific) at 530 nm excitation/620 nm emission.

    Techniques: Expressing, Functional Assay, Western Blot, Positive Control, Immunofluorescence, Incubation, Negative Control

    Akt requires a hydrolyzable glucose substrate to prevent death upon growth factor withdrawal. Control (Neo) (A) and mAkt-transfected IL-3-dependent FL5.12 (B) cells were withdrawn from IL-3 in the absence of glucose or in the presence of 10 mM glucose or 10 mM 2-DOG, a glucose analog that cannot be metabolized through glycolysis. At various time points, cells were analyzed by flow cytometry to determine viability by propidium iodide exclusion. (C) The glycolytic rates of Neo and mAkt cells were measured after 10 h in the absence of IL-3. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.

    Journal: Molecular and Cellular Biology

    Article Title: Akt-Directed Glucose Metabolism Can Prevent Bax Conformation Change and Promote Growth Factor-Independent Survival

    doi: 10.1128/MCB.23.20.7315-7328.2003

    Figure Lengend Snippet: Akt requires a hydrolyzable glucose substrate to prevent death upon growth factor withdrawal. Control (Neo) (A) and mAkt-transfected IL-3-dependent FL5.12 (B) cells were withdrawn from IL-3 in the absence of glucose or in the presence of 10 mM glucose or 10 mM 2-DOG, a glucose analog that cannot be metabolized through glycolysis. At various time points, cells were analyzed by flow cytometry to determine viability by propidium iodide exclusion. (C) The glycolytic rates of Neo and mAkt cells were measured after 10 h in the absence of IL-3. The values shown are the means of the results for triplicate samples; error bars indicate standard deviations.

    Article Snippet: To determine cell viability, cells were stained with 10 μg of the vital dye propidium iodide (PI; Molecular Probes, Eugene, Oreg.) per ml.

    Techniques: Transfection, Flow Cytometry, Cytometry

    Coexpression of Glut1 and HK1 increases survival in the absence of IL-3 and prevents change in Bax conformation. (A) Cells were withdrawn from IL-3, and viabilities at various time points were measured by flow cytometry by propidium iodide exclusion. The values shown are the means of the results for cell survival from triplicate samples; error bars indicate standard deviations. (B) Bax activation was measured by flow cytometry in cells that were maintained in IL-3 or withdrawn from IL-3 for 12 h. Percentages for cells staining positive for change in Bax conformation are given. (C) Control (Neo) and mAkt-expressing cells were cultured in the presence or absence of glucose or IL-3, as indicated, for 12 h, and the percentage of cells with activated Bax was determined. (D) Control (a), Glut1/HK1 (b), mAkt (c), and Bcl-xL (d)-overexpressing FL5.12 cells were cultured in the absence of IL-3 either with (solid lines) or without (dashed lines) the glycolytic inhibitor IAA. Cell viabilities at various time points after the start of culture were measured by flow cytometry by propidium iodide exclusion. The values shown are the means for the results of cell survival from triplicate samples; error bars indicate standard deviations.

    Journal: Molecular and Cellular Biology

    Article Title: Akt-Directed Glucose Metabolism Can Prevent Bax Conformation Change and Promote Growth Factor-Independent Survival

    doi: 10.1128/MCB.23.20.7315-7328.2003

    Figure Lengend Snippet: Coexpression of Glut1 and HK1 increases survival in the absence of IL-3 and prevents change in Bax conformation. (A) Cells were withdrawn from IL-3, and viabilities at various time points were measured by flow cytometry by propidium iodide exclusion. The values shown are the means of the results for cell survival from triplicate samples; error bars indicate standard deviations. (B) Bax activation was measured by flow cytometry in cells that were maintained in IL-3 or withdrawn from IL-3 for 12 h. Percentages for cells staining positive for change in Bax conformation are given. (C) Control (Neo) and mAkt-expressing cells were cultured in the presence or absence of glucose or IL-3, as indicated, for 12 h, and the percentage of cells with activated Bax was determined. (D) Control (a), Glut1/HK1 (b), mAkt (c), and Bcl-xL (d)-overexpressing FL5.12 cells were cultured in the absence of IL-3 either with (solid lines) or without (dashed lines) the glycolytic inhibitor IAA. Cell viabilities at various time points after the start of culture were measured by flow cytometry by propidium iodide exclusion. The values shown are the means for the results of cell survival from triplicate samples; error bars indicate standard deviations.

    Article Snippet: To determine cell viability, cells were stained with 10 μg of the vital dye propidium iodide (PI; Molecular Probes, Eugene, Oreg.) per ml.

    Techniques: Flow Cytometry, Cytometry, Activation Assay, Staining, Expressing, Cell Culture

    Percentage of dead CSM 14.1 and iBMK cells assessed by propidium iodide exclusion after 24 h of treatment with 1 μ M staurosporine. Both cell types were stably transfected with YFP, YFP-BCL-x L , YFP-BCL-x L -ΔTM, or YFP-TM. Mean ±

    Journal:

    Article Title: The C-Terminal Transmembrane Domain of Bcl-xL Mediates Changes in Mitochondrial Morphology

    doi: 10.1529/biophysj.107.104323

    Figure Lengend Snippet: Percentage of dead CSM 14.1 and iBMK cells assessed by propidium iodide exclusion after 24 h of treatment with 1 μ M staurosporine. Both cell types were stably transfected with YFP, YFP-BCL-x L , YFP-BCL-x L -ΔTM, or YFP-TM. Mean ±

    Article Snippet: After 24 h, 40 μ M propidium iodide (Invitrogen) was added to the incubated cultures for 15 min.

    Techniques: Stable Transfection, Transfection

    CLSM micrographs comparing biofilm formation of GBS 10/84 grown in THB supplemented with 1% glucose or THB supplemented with 1% glucose and HMOs isolated from milk donors. Bacteria were grown under static conditions at 37°C for 24 hours on glass coverslips. Biofilms were stained immediately prior to analysis with SYTO-9 (green, live bacterial cells), propidium iodide (red, dead bacterial cells), and Calcofluor White (blue, carbohydrates) at 600× magnification. Images shown represent a z-stack series of images of the three stains where the larger panel is a “bird´s eye” view of the biofilms and the right and upper panels are side views of the x- and y-axis sections respectively.

    Journal: ACS infectious diseases

    Article Title: Human Milk Oligosaccharides Exhibit Antimicrobial and Anti-Biofilm Properties Against Group B Streptococcus

    doi: 10.1021/acsinfecdis.7b00064

    Figure Lengend Snippet: CLSM micrographs comparing biofilm formation of GBS 10/84 grown in THB supplemented with 1% glucose or THB supplemented with 1% glucose and HMOs isolated from milk donors. Bacteria were grown under static conditions at 37°C for 24 hours on glass coverslips. Biofilms were stained immediately prior to analysis with SYTO-9 (green, live bacterial cells), propidium iodide (red, dead bacterial cells), and Calcofluor White (blue, carbohydrates) at 600× magnification. Images shown represent a z-stack series of images of the three stains where the larger panel is a “bird´s eye” view of the biofilms and the right and upper panels are side views of the x- and y-axis sections respectively.

    Article Snippet: At 24 hours, coverslips were washed with PBS prior to staining with LIVE/DEAD BacLight bacterial viability kit, which includes both Syto 9 (green) and propidium iodide (red) (Life Technologies) to visualize bacterial cells and calcofluor white (blue) (Sigma-Aldrich) to visualize carbohydrate capsule/matrix within the biofilm.

    Techniques: Confocal Laser Scanning Microscopy, Isolation, Staining

    Images of Live/Dead stained biofilms. Biofilms were incubated on coupons for 24 hours before staining with Syto9 and propidium iodide. Images were taken with an Olympus IX83 fluorescence microscope using CellSense software. ( A ) B. subtilis , ( B ) P. aeruginosa , ( C ) K. carniphila , ( D ) P. libanensis , ( E ) S. epidermidis , ( F ) E. cloacae .

    Journal: Scientific Reports

    Article Title: Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma

    doi: 10.1038/srep38610

    Figure Lengend Snippet: Images of Live/Dead stained biofilms. Biofilms were incubated on coupons for 24 hours before staining with Syto9 and propidium iodide. Images were taken with an Olympus IX83 fluorescence microscope using CellSense software. ( A ) B. subtilis , ( B ) P. aeruginosa , ( C ) K. carniphila , ( D ) P. libanensis , ( E ) S. epidermidis , ( F ) E. cloacae .

    Article Snippet: Biofilm imaging To visualize the biofilms, cells were stained by covering the coupon with 100 μl of the BacLight Live/dead stain containing the 2 stock solutions green Syto 9 and red propidium iodide (Molecular Probes, Eugene, Oregon, USA) according to the manufacturer’s instructions.

    Techniques: Staining, Incubation, Fluorescence, Microscopy, Software

    Flow cytometry analysis of cell lines grown in low serum condition with or without addition of NT-3. Annexin-V/propidium iodide double staining was performed to determine the rate of early apoptosis (Q4) and apoptotic cells (Q2). The data shown is representative for results in cell lines expressing WT TrkC, TrkC p.T93M, and TrkC p.N163I on day 7.

    Journal: Human mutation

    Article Title: Mutations in NTRK3 suggest a novel signaling pathway in human congenital heart disease

    doi: 10.1002/humu.22688

    Figure Lengend Snippet: Flow cytometry analysis of cell lines grown in low serum condition with or without addition of NT-3. Annexin-V/propidium iodide double staining was performed to determine the rate of early apoptosis (Q4) and apoptotic cells (Q2). The data shown is representative for results in cell lines expressing WT TrkC, TrkC p.T93M, and TrkC p.N163I on day 7.

    Article Snippet: The Dead Cell Apoptosis Kit with Annexin V alexa Fluor 488 & Propidium Iodide kit (Life Technologies, Eugene, OR) was used to stain the cells.

    Techniques: Flow Cytometry, Cytometry, Double Staining, Expressing

    Brainstem slice cultures are viable. An individual single necrotic cells (8 ± 3%, n=257, propidium iodide-stained) were found in the brainstem slice cultures (N=12) that had been cultivated for 3 weeks, but no large clusters of necrotic cells were detected ( a , d ). The few necrotic cells were observed in the thickest regions, indicating that diffusion-based oxygenation is critically dependent on slice thickness. Oxygen glucose deprivation (OGD) for 1 hr produced clear positive PI-staining throughout the slice ( b ; N=5). Cultures also showed low apoptotic activity (2 ± 1%, n=187), as evaluated by caspase-3 staining ( c , d ; N=20). DIV: days in vitro. N: slices, n: cells. Scale bars: 100 µm. DOI: http://dx.doi.org/10.7554/eLife.14170.010

    Journal: eLife

    Article Title: CO2-evoked release of PGE2 modulates sighs and inspiration as demonstrated in brainstem organotypic culture

    doi: 10.7554/eLife.14170

    Figure Lengend Snippet: Brainstem slice cultures are viable. An individual single necrotic cells (8 ± 3%, n=257, propidium iodide-stained) were found in the brainstem slice cultures (N=12) that had been cultivated for 3 weeks, but no large clusters of necrotic cells were detected ( a , d ). The few necrotic cells were observed in the thickest regions, indicating that diffusion-based oxygenation is critically dependent on slice thickness. Oxygen glucose deprivation (OGD) for 1 hr produced clear positive PI-staining throughout the slice ( b ; N=5). Cultures also showed low apoptotic activity (2 ± 1%, n=187), as evaluated by caspase-3 staining ( c , d ; N=20). DIV: days in vitro. N: slices, n: cells. Scale bars: 100 µm. DOI: http://dx.doi.org/10.7554/eLife.14170.010

    Article Snippet: Propidium iodide staining Propidium iodide (1 ml/L, Invitrogen, UK) was added to brain slice medium (dilution 1:1000).

    Techniques: Staining, Diffusion-based Assay, Produced, Activity Assay, In Vitro

    Microscopy images of mitoxantrone treated PC3-TR cells . Left panels are phase contrast images and right panels are fluorescence images visualized with an annexin V and propidium iodide stain. Apoptotic cells exhibit green fluorescence only. Dead cells can exhibit either lone red fluorescence or red and green fluorescence simultaneously. Live cells are non-fluorescent. Arrows identify apoptotic cells.

    Journal: BMC Cancer

    Article Title: Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells

    doi: 10.1186/1471-2407-11-470

    Figure Lengend Snippet: Microscopy images of mitoxantrone treated PC3-TR cells . Left panels are phase contrast images and right panels are fluorescence images visualized with an annexin V and propidium iodide stain. Apoptotic cells exhibit green fluorescence only. Dead cells can exhibit either lone red fluorescence or red and green fluorescence simultaneously. Live cells are non-fluorescent. Arrows identify apoptotic cells.

    Article Snippet: Annexin V and Propidium Iodide Analysis An annexin V/propidium iodide assay (Invitrogen L3224) was carried out to determine if combination treatments induced apoptosis in cells.

    Techniques: Microscopy, Fluorescence, Staining

    Cell death of Salmonella -infected THP1 cells is determined by PMA concentration. (A) Representative bright field images of THP-1 cells differentiated for 3 days in the presence of 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA, as described in Material and Methods. Scale bar is 100 μm. (B) Quantification of percentage of propidium iodide positive THP-1 cells differentiated for 2 days, 3 days, and 4 days at either 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA following infection with Salmonella (filled circle) for 2 h as compared to uninfected (open circles). Data are means ± SD from three independent experiments. Statistical significance was determined using 2-way ANOVA with Sidak post hoc test. (C) Representative images of differentiated THP-1 cells stained with propidium iodide (red) 2 h pi with Salmonella (bottom panel) or uninfected (top panel). Scale bar 25μm.

    Journal: PLoS ONE

    Article Title: The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium

    doi: 10.1371/journal.pone.0193601

    Figure Lengend Snippet: Cell death of Salmonella -infected THP1 cells is determined by PMA concentration. (A) Representative bright field images of THP-1 cells differentiated for 3 days in the presence of 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA, as described in Material and Methods. Scale bar is 100 μm. (B) Quantification of percentage of propidium iodide positive THP-1 cells differentiated for 2 days, 3 days, and 4 days at either 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA following infection with Salmonella (filled circle) for 2 h as compared to uninfected (open circles). Data are means ± SD from three independent experiments. Statistical significance was determined using 2-way ANOVA with Sidak post hoc test. (C) Representative images of differentiated THP-1 cells stained with propidium iodide (red) 2 h pi with Salmonella (bottom panel) or uninfected (top panel). Scale bar 25μm.

    Article Snippet: Where indicated cells were treated with LPS from Salmonella enterica serotype Minnesota (10 ng/mL, Sigma) for 30 min. For propidium iodide assays, cells were incubated with propidium iodide (1 μg/mL, Life Technologies) for 15 minutes at the appropriate time post-infection and immediately analyzed by fluorescence microscopy without fixation.

    Techniques: Infection, Concentration Assay, Staining

    LPS induced cell death in differentiated THP-1 cells. (A) Quantification of percentage of propidium iodide positive THP-1 cells differentiated in the presence of PMA (200 ng/mL) for 2 days (white circles) or 5 days (black circles) following internalization of Salmonella or E . coli DH5α or a 30 min treatment with LPS (10 ng/mL). Data shown as individual experimental means. Statistical significance was determined as compared to uninfected controls using 2-way ANOVA with Tukey’s post hoc test. (B) Representative bright field images of THP-1 cells stained with propidium iodide (red) 120 min after internalization of Salmonella or E . coli or treatment with LPS. Scale bar 15 μm. (C) THP-1 cells were treated with DMSO or staurosporine (1 μM) for 3 h, infected with Salmonella or E . Coli for 30 min, or treated with LPS (10 ng/mL) for 30 min. At 2 h post-treatment cells were incubated with poly-caspase FAM-VAD-FMK before fixation, staining, and analysis. Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.

    Journal: PLoS ONE

    Article Title: The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium

    doi: 10.1371/journal.pone.0193601

    Figure Lengend Snippet: LPS induced cell death in differentiated THP-1 cells. (A) Quantification of percentage of propidium iodide positive THP-1 cells differentiated in the presence of PMA (200 ng/mL) for 2 days (white circles) or 5 days (black circles) following internalization of Salmonella or E . coli DH5α or a 30 min treatment with LPS (10 ng/mL). Data shown as individual experimental means. Statistical significance was determined as compared to uninfected controls using 2-way ANOVA with Tukey’s post hoc test. (B) Representative bright field images of THP-1 cells stained with propidium iodide (red) 120 min after internalization of Salmonella or E . coli or treatment with LPS. Scale bar 15 μm. (C) THP-1 cells were treated with DMSO or staurosporine (1 μM) for 3 h, infected with Salmonella or E . Coli for 30 min, or treated with LPS (10 ng/mL) for 30 min. At 2 h post-treatment cells were incubated with poly-caspase FAM-VAD-FMK before fixation, staining, and analysis. Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.

    Article Snippet: Where indicated cells were treated with LPS from Salmonella enterica serotype Minnesota (10 ng/mL, Sigma) for 30 min. For propidium iodide assays, cells were incubated with propidium iodide (1 μg/mL, Life Technologies) for 15 minutes at the appropriate time post-infection and immediately analyzed by fluorescence microscopy without fixation.

    Techniques: Staining, Infection, Incubation

    Ngζ_1 expression in E. coli cells causes a lytic phenotype compensated by ngε_1 co-expression. a Growth curves of E. coli C41(DE3) cells expressing ngζ_1 (red solid line and left Y -axis) or ngζ_1(K115A) (forest-green solid line and left Y -axis) together with the corresponding propidium iodide influx into the cells (dashed lines similarly colored and right Y -axis). Control experiments in which expression of ngζ_1 or ngζ_1(K115A) was not induced are shown in light pink and lime-green. Note that growth curves of the two uninduced cell cultures as well as the PI measurements of the mutated variant (induced and not induced) fully overlay with each other. b Growth curves of E. coli BL21(DE3)-RIL cells co-expressing ngζ_1 and ngε_1. Expression of ngε_1 was induced 0 (forest-green), 15 (lime-green) and 30 min (pale-green) subsequent to ngζ_1 induction. The control experiment in which expression of ngε_1 was not induced is shown in red. c Phase contrast and fluorescence microscopy (live-dead stain) of E. coli C41(DE3) cells before and after ngζ_1 induction (30 min) and the control experiment using the inactive ngζ_1(K115A) mutant. Cells with green fluorescence have intact cell membranes and are alive. Membrane permeable cells have red fluorescence due to propidium iodide influx. Resolved bulges due to cell wall extrusions in phase-contrast microscopy images are highlighted with red arrows

    Journal: Nature Communications

    Article Title: The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis

    doi: 10.1038/s41467-018-03652-8

    Figure Lengend Snippet: Ngζ_1 expression in E. coli cells causes a lytic phenotype compensated by ngε_1 co-expression. a Growth curves of E. coli C41(DE3) cells expressing ngζ_1 (red solid line and left Y -axis) or ngζ_1(K115A) (forest-green solid line and left Y -axis) together with the corresponding propidium iodide influx into the cells (dashed lines similarly colored and right Y -axis). Control experiments in which expression of ngζ_1 or ngζ_1(K115A) was not induced are shown in light pink and lime-green. Note that growth curves of the two uninduced cell cultures as well as the PI measurements of the mutated variant (induced and not induced) fully overlay with each other. b Growth curves of E. coli BL21(DE3)-RIL cells co-expressing ngζ_1 and ngε_1. Expression of ngε_1 was induced 0 (forest-green), 15 (lime-green) and 30 min (pale-green) subsequent to ngζ_1 induction. The control experiment in which expression of ngε_1 was not induced is shown in red. c Phase contrast and fluorescence microscopy (live-dead stain) of E. coli C41(DE3) cells before and after ngζ_1 induction (30 min) and the control experiment using the inactive ngζ_1(K115A) mutant. Cells with green fluorescence have intact cell membranes and are alive. Membrane permeable cells have red fluorescence due to propidium iodide influx. Resolved bulges due to cell wall extrusions in phase-contrast microscopy images are highlighted with red arrows

    Article Snippet: Two hundred microliter of these mixtures were transferred into a 96-well plate (Corning 3651, Corning Inc, Corning, USA) and OD600 and propidium iodide fluorescence (excitation: 520 nm, emission: 620 nm) was monitored over 90 min using a Varioscan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, USA).

    Techniques: Expressing, Variant Assay, Fluorescence, Microscopy, Staining, Mutagenesis

    A–G) Frontal views of the buccopharyngeal membrane labeled with pJNK1 antibody (green) at three different stages representing prior to and during perforation. White dots outline “holes” or perforations. H) Sagittal section through the buccopharyngeal membrane and surrounding face showing pJNK (green) and counterstained with propidium iodide. pJNK can also be observed in the two layered epidermis, in the outer epidermis (OE) and inner epidermis (IE). I) Flat mount of the epidermis labeled with pJNK antibody (green). J) The mesoderm labeled with pJNK antibody (green). J′) Corresponding image to J where the pJNK is labeled green and nuclei/DNA labeled with propidium iodide (red). White arrows indicate examples of cells that appear to be undergoing mitosis. K) Magnified image of a mesodermal cell labeled with pJNK antibody (green) K′) corresponding cell from K labeled with propidium iodide to show DNA (red). K″) Cell corresponding to K showing a merge of pJNK (green) and DNA (red). L) Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged control morpholino (green). L′) Same image as L showing only the pJNK labeling (red). M) Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged JNK1 morpholino (green) M′) same image as H showing only the pJNK labeling (red). Abbreviations: pJNK1=phospho JNK1, st. =stage.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: The role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation

    doi: 10.1002/dvdy.24470

    Figure Lengend Snippet: A–G) Frontal views of the buccopharyngeal membrane labeled with pJNK1 antibody (green) at three different stages representing prior to and during perforation. White dots outline “holes” or perforations. H) Sagittal section through the buccopharyngeal membrane and surrounding face showing pJNK (green) and counterstained with propidium iodide. pJNK can also be observed in the two layered epidermis, in the outer epidermis (OE) and inner epidermis (IE). I) Flat mount of the epidermis labeled with pJNK antibody (green). J) The mesoderm labeled with pJNK antibody (green). J′) Corresponding image to J where the pJNK is labeled green and nuclei/DNA labeled with propidium iodide (red). White arrows indicate examples of cells that appear to be undergoing mitosis. K) Magnified image of a mesodermal cell labeled with pJNK antibody (green) K′) corresponding cell from K labeled with propidium iodide to show DNA (red). K″) Cell corresponding to K showing a merge of pJNK (green) and DNA (red). L) Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged control morpholino (green). L′) Same image as L showing only the pJNK labeling (red). M) Embryos labeled with pJNK antibody (red) and showing the fluorescein tagged JNK1 morpholino (green) M′) same image as H showing only the pJNK labeling (red). Abbreviations: pJNK1=phospho JNK1, st. =stage.

    Article Snippet: In some cases propidium iodide (1:1000 for 1 hour at room temperature) was used as a counterstain as Mitotracker Red CMXRos (ThermoFisher, M7512) was used in live embryos.

    Techniques: Labeling

    A,B) Lateral view of representative controls (A) and JNK1 morphants (B) where gst1 mRNA (purple) was detected by in situ hybridization. C–F) Live Mitotracker CMXRos labeling (red) after embryos were treated with DMSO (control; C, E) or JNK inhibitor. C, D) Shows the buccopharyngeal membrane. E, F) Shows the epidermis. G, H) Frontal views of the face of representative embryos untreated (G) or treated with hydrogen peroxide (H202; H). Red dots outline the embryonic mouth. I, J) Transverse sections through the face of a representative embryo injected with JNK1 morpholino into one cell at the two cell stage. Tissue containing the fluorescein tagged JNK1 morpholino is green and cleaved caspase-3 labeling (apoptotic marker) is in red. K, L) Frontal views of whole representative faces from embryos treated with DMSO (K) or JNK inhibitor (L). Cleaved caspase-3 labeling is in green and propidium iodide is used a counterstain (red). M, N) Representative images of frontal views of faces of embryos exposed to UV. M) Control (not irradiated) and N) Irradiated at stage 37/38. Red dots outline the embryonic mouth. Abbreviations; perf. =perforated, MO= morpholino, BM = buccopharyngeal membrane, CC3=cleaved caspase 3, PI= propidium iodide.

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: The role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation

    doi: 10.1002/dvdy.24470

    Figure Lengend Snippet: A,B) Lateral view of representative controls (A) and JNK1 morphants (B) where gst1 mRNA (purple) was detected by in situ hybridization. C–F) Live Mitotracker CMXRos labeling (red) after embryos were treated with DMSO (control; C, E) or JNK inhibitor. C, D) Shows the buccopharyngeal membrane. E, F) Shows the epidermis. G, H) Frontal views of the face of representative embryos untreated (G) or treated with hydrogen peroxide (H202; H). Red dots outline the embryonic mouth. I, J) Transverse sections through the face of a representative embryo injected with JNK1 morpholino into one cell at the two cell stage. Tissue containing the fluorescein tagged JNK1 morpholino is green and cleaved caspase-3 labeling (apoptotic marker) is in red. K, L) Frontal views of whole representative faces from embryos treated with DMSO (K) or JNK inhibitor (L). Cleaved caspase-3 labeling is in green and propidium iodide is used a counterstain (red). M, N) Representative images of frontal views of faces of embryos exposed to UV. M) Control (not irradiated) and N) Irradiated at stage 37/38. Red dots outline the embryonic mouth. Abbreviations; perf. =perforated, MO= morpholino, BM = buccopharyngeal membrane, CC3=cleaved caspase 3, PI= propidium iodide.

    Article Snippet: In some cases propidium iodide (1:1000 for 1 hour at room temperature) was used as a counterstain as Mitotracker Red CMXRos (ThermoFisher, M7512) was used in live embryos.

    Techniques: In Situ Hybridization, Labeling, Injection, Marker, Irradiation

    The design ( left ) and results ( right ) of the retrograde tracing experiment. FB injections into the olfactory bulb ( OB ) and piriform cortex ( PO ) were implemented to define projection targets of dying cortical neurons. Axotomized neurons projecting to the olfactory bulb ( top neuron in the diagram ) remain intact after the lesion. Nonaxotomized neurons projecting within piriform cortex ( bottom neuron in A , corresponding to neurons in A–C ) undergo apoptosis. A , Portion of the olfactory cortex of a bulbectomized rat retrogradely labeled with FB injected into the olfactory bulb before the lesion and counterstained with propidium iodide. The animal survived for 24 hr. FB labels neurons in layer IIb and does not colocalize in apoptotic cells ( arrows indicate an apoptotic neuron). B, C , Two apoptotic profiles ( arrows ) labeled postinjection of FB into the posterior piriform cortex. Fluorescent labeling shows that they are projection (pyramidal) neurons. The fact that these neurons cannot be labeled postinjection into olfactory bulb but become apoptotic postbulbectomy indicates that they are trans-synaptically affected by the lesion. Arrowheads in B show the axon of an apoptotic profile undergoing Wallerian degeneration. Scale bars, 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Deafferentation Causes Apoptosis in Cortical Sensory Neurons in the Adult Rat

    doi: 10.1523/JNEUROSCI.17-19-07372.1997

    Figure Lengend Snippet: The design ( left ) and results ( right ) of the retrograde tracing experiment. FB injections into the olfactory bulb ( OB ) and piriform cortex ( PO ) were implemented to define projection targets of dying cortical neurons. Axotomized neurons projecting to the olfactory bulb ( top neuron in the diagram ) remain intact after the lesion. Nonaxotomized neurons projecting within piriform cortex ( bottom neuron in A , corresponding to neurons in A–C ) undergo apoptosis. A , Portion of the olfactory cortex of a bulbectomized rat retrogradely labeled with FB injected into the olfactory bulb before the lesion and counterstained with propidium iodide. The animal survived for 24 hr. FB labels neurons in layer IIb and does not colocalize in apoptotic cells ( arrows indicate an apoptotic neuron). B, C , Two apoptotic profiles ( arrows ) labeled postinjection of FB into the posterior piriform cortex. Fluorescent labeling shows that they are projection (pyramidal) neurons. The fact that these neurons cannot be labeled postinjection into olfactory bulb but become apoptotic postbulbectomy indicates that they are trans-synaptically affected by the lesion. Arrowheads in B show the axon of an apoptotic profile undergoing Wallerian degeneration. Scale bars, 10 μm.

    Article Snippet: Frozen sections (20 μm) were cut on a cryostat at the coronal plane through the rostral half of piriform cortex, dry-mounted on gelatin-subbed slides, and counterstained with 0.001% aqueous propidium iodide (Molecular Probes, Eugene, OR; used as an in situ DNA marker).

    Techniques: Retrograde Tracing, Labeling, Injection

    Effect of EGCG on cell death . Fluorescent images representing the effect of increasing concentrations of EGCG on cell death. The fluorescent probe propidium iodide was used to visualize dead cells. Compared to control (A) or to 5 μg/ml (B) and 10 μg/ml (C) of EGCG, we observed that 20 μg/ml (D) of EGCG induced a significant (p

    Journal: Respiratory Research

    Article Title: Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

    doi: 10.1186/1465-9921-9-33

    Figure Lengend Snippet: Effect of EGCG on cell death . Fluorescent images representing the effect of increasing concentrations of EGCG on cell death. The fluorescent probe propidium iodide was used to visualize dead cells. Compared to control (A) or to 5 μg/ml (B) and 10 μg/ml (C) of EGCG, we observed that 20 μg/ml (D) of EGCG induced a significant (p

    Article Snippet: After 18 h of cell interaction with EGCG, the fluorescent probe propidium iodide (Invitrogen, Cergy Pontoise, France), diluted at 20 mM in the culture medium, was used to visualize the cell death.

    Techniques:

    3D projections of biofilms obtained from confocal microscopy data ( imaris software). Blue: exopolysaccharides matrix stained with concanavalin A conjugated to tetramethylrhodamine. Green: bacterial cells stained with SYTO 9. Red: dead cells stained with propidium iodide. Scale bar: 3.10 4 μm. A. 24 h. Microcolonies and many dense polysaccharide aggregates were observed, even after this short time of incubation. B. 72 h. The biovolume of cells was more important for Tm . sp. CB 2, and these cells cover uniformly the surface. A high proportion of dead cells was observed in the biofilm made by Tm . sp. CB 1, as compared to the other strains.

    Journal: Microbial Biotechnology

    Article Title: Comparison of biofilm formation and motility processes in arsenic‐resistant Thiomonas spp. strains revealed divergent response to arsenite

    doi: 10.1111/1751-7915.12556

    Figure Lengend Snippet: 3D projections of biofilms obtained from confocal microscopy data ( imaris software). Blue: exopolysaccharides matrix stained with concanavalin A conjugated to tetramethylrhodamine. Green: bacterial cells stained with SYTO 9. Red: dead cells stained with propidium iodide. Scale bar: 3.10 4 μm. A. 24 h. Microcolonies and many dense polysaccharide aggregates were observed, even after this short time of incubation. B. 72 h. The biovolume of cells was more important for Tm . sp. CB 2, and these cells cover uniformly the surface. A high proportion of dead cells was observed in the biofilm made by Tm . sp. CB 1, as compared to the other strains.

    Article Snippet: Dead cells were observed after propidium iodide labelling (Life Technologies); however, the volumes of dead cells could hardly be quantified with accuracy as the labelling with propidium iodide gave a high background noise in those samples.

    Techniques: Confocal Microscopy, Software, Staining, Incubation

    Penetration of CIGB-210 into the MT4 cell line. ( A , B ) CIGB-210 uptake assessed by flow cytometry. MT4 cells were incubated with 10, 20 or 40 µM of carboxyfluorescein labeled CIGB-210 for 15 min, 60 min or 24 h. External fluorescence was quenched by addition of 0.4% Trypan blue. CC: Control untreated MT4 cells; PP: carboxyfluorescein labeled peptide containing the Tat cell penetrating peptide used as positive control; ( A ) Flow cytometry histograms; and ( B ) percentage of fluorescent cells for each experimental condition. Data represent the mean ± standard deviation of three experiments performed in triplicate; ( C – E ) CIGB-210 uptake assessed by fluorescence microscopy. MT4 cells were treated with: 10 µM biotinylated CIGB-210 ( E ); 10 µM biotinylated Tat cell penetrating peptide ( D ); or medium ( C ) for 24 h, fixed and visualized with a streptavidin-FITC conjugate. The nucleus was stained with propidium iodine ( red ). 100× magnification.

    Journal: Viruses

    Article Title: Identification of Vimentin as a Potential Therapeutic Target against HIV Infection

    doi: 10.3390/v8060098

    Figure Lengend Snippet: Penetration of CIGB-210 into the MT4 cell line. ( A , B ) CIGB-210 uptake assessed by flow cytometry. MT4 cells were incubated with 10, 20 or 40 µM of carboxyfluorescein labeled CIGB-210 for 15 min, 60 min or 24 h. External fluorescence was quenched by addition of 0.4% Trypan blue. CC: Control untreated MT4 cells; PP: carboxyfluorescein labeled peptide containing the Tat cell penetrating peptide used as positive control; ( A ) Flow cytometry histograms; and ( B ) percentage of fluorescent cells for each experimental condition. Data represent the mean ± standard deviation of three experiments performed in triplicate; ( C – E ) CIGB-210 uptake assessed by fluorescence microscopy. MT4 cells were treated with: 10 µM biotinylated CIGB-210 ( E ); 10 µM biotinylated Tat cell penetrating peptide ( D ); or medium ( C ) for 24 h, fixed and visualized with a streptavidin-FITC conjugate. The nucleus was stained with propidium iodine ( red ). 100× magnification.

    Article Snippet: The slides were washed three times with PBS, treated for 30 s with propidium iodine (Sigma-Aldrich, USA) at 10 µg/mL and washed again three times as described before.

    Techniques: Flow Cytometry, Cytometry, Incubation, Labeling, Fluorescence, Positive Control, Standard Deviation, Microscopy, Staining

    Immunofluorescence microphotographs of vimentin IFs in different cellular contexts: ( A ) MT4 cells; ( B ) MT4 cells treated with the B1 fraction; ( C ) MT4mock cells; and ( D ) MT4sh/Vim cells. Cells were fixed and vimentin IFs were visualized with an anti-vimentin mouse monoclonal antibody followed by a FITC conjugated anti-mouse IgG antibody ( green ). The nucleus was stained with propidium iodine ( red ). The localization of vimentin IFs in ( A ) and ( C ) was mostly delimited to one edge of the cell ( A ) 264 cells out of 291 for a 90.7%; and ( C ) 260 cells out of 323 for an 80.4%). In contrast, a redistribution in vimentin IFs was observed in ( B ) (226 cells out of 311 for a 72%). Scarce, scattered and unpolarized vimentin filaments were observed in MT4sh/Vim cells ( D ) 190 cells out of 207 for a 91.7%). 40× magnification.

    Journal: Viruses

    Article Title: Identification of Vimentin as a Potential Therapeutic Target against HIV Infection

    doi: 10.3390/v8060098

    Figure Lengend Snippet: Immunofluorescence microphotographs of vimentin IFs in different cellular contexts: ( A ) MT4 cells; ( B ) MT4 cells treated with the B1 fraction; ( C ) MT4mock cells; and ( D ) MT4sh/Vim cells. Cells were fixed and vimentin IFs were visualized with an anti-vimentin mouse monoclonal antibody followed by a FITC conjugated anti-mouse IgG antibody ( green ). The nucleus was stained with propidium iodine ( red ). The localization of vimentin IFs in ( A ) and ( C ) was mostly delimited to one edge of the cell ( A ) 264 cells out of 291 for a 90.7%; and ( C ) 260 cells out of 323 for an 80.4%). In contrast, a redistribution in vimentin IFs was observed in ( B ) (226 cells out of 311 for a 72%). Scarce, scattered and unpolarized vimentin filaments were observed in MT4sh/Vim cells ( D ) 190 cells out of 207 for a 91.7%). 40× magnification.

    Article Snippet: The slides were washed three times with PBS, treated for 30 s with propidium iodine (Sigma-Aldrich, USA) at 10 µg/mL and washed again three times as described before.

    Techniques: Immunofluorescence, Staining

    Lovastatin induces downregulation of the Skp2 protein (A) HeLa cells growing asynchronously or arrested in S-Phase by treatment with 2mM thymidine for 24 hours, were then treated or left untreated with 40μM lovastatin for a further 24 hours and analysed by (A) immunoblotting. (B) LnCAP and HeLa cells (C) were incubated with 10μM and 40μM lovastatin respectively for the indicated times and analysed by immunoblotting. (D) HeLa cells stably expressing Skp2 from a PGK promoter were generated by lentiviral transfection. These cells were treated with 40μM lovastatin for 24 hours and then subjected to immunoblotting analysis. Percentages of cells in the different cell cycle phases were determined by flow cytometric analysis of three independent experiments using propidium iodine and BrdU double staining (Bar graph; data are represented as mean +/− SEM). (E) Control MEFs and p27 −/− MEFs were treated with 20μM lovastatin for 24 hours as indicated, harvested and analysed by western blotting analysis (left panel). Flow cytometry analysis of BrdU labeled cells to determine cell cycle phase distribution was performed. The percentage of cells in G1, S or G2/M phase is shown for at least three independent experiments (right panel). (F) Skp2 mRNA analysis of HeLa cells treated as described in Fig. 1A . Northern blots from three independent experiments were quantified using a PhosphorImager and the relative abundance of the Skp2 mRNA is shown (bar graph; data are represented as mean +/− SEM).

    Journal: Oncotarget

    Article Title: Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation

    doi:

    Figure Lengend Snippet: Lovastatin induces downregulation of the Skp2 protein (A) HeLa cells growing asynchronously or arrested in S-Phase by treatment with 2mM thymidine for 24 hours, were then treated or left untreated with 40μM lovastatin for a further 24 hours and analysed by (A) immunoblotting. (B) LnCAP and HeLa cells (C) were incubated with 10μM and 40μM lovastatin respectively for the indicated times and analysed by immunoblotting. (D) HeLa cells stably expressing Skp2 from a PGK promoter were generated by lentiviral transfection. These cells were treated with 40μM lovastatin for 24 hours and then subjected to immunoblotting analysis. Percentages of cells in the different cell cycle phases were determined by flow cytometric analysis of three independent experiments using propidium iodine and BrdU double staining (Bar graph; data are represented as mean +/− SEM). (E) Control MEFs and p27 −/− MEFs were treated with 20μM lovastatin for 24 hours as indicated, harvested and analysed by western blotting analysis (left panel). Flow cytometry analysis of BrdU labeled cells to determine cell cycle phase distribution was performed. The percentage of cells in G1, S or G2/M phase is shown for at least three independent experiments (right panel). (F) Skp2 mRNA analysis of HeLa cells treated as described in Fig. 1A . Northern blots from three independent experiments were quantified using a PhosphorImager and the relative abundance of the Skp2 mRNA is shown (bar graph; data are represented as mean +/− SEM).

    Article Snippet: FTI-277 (Sigma-Aldrich #F9803), BrdU (Sigma-Aldrich #B9285), propidium iodine (PI) (Sigma-Aldrich #81845).

    Techniques: Incubation, Stable Transfection, Expressing, Generated, Transfection, Flow Cytometry, Double Staining, Western Blot, Cytometry, Labeling, Northern Blot

    CEP2 loss of function results in shortening the cell length and cell width of the longest trichoblasts (double arrows) in the root elongation zone at the level of the transition from the rapid elongation zone to the late elongation zone. The residual CEP2 activity in cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) results in prolonged trichoblasts as compared to cep2 ko mutant plants, but shorter as compared to trichoblasts in WT plants. (A) 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd plants were stained with propidium iodide and analyzed by CLSM (single pictures, 200-fold magnification). Two examples of propidium iodide stained roots are presented for each genotype. (B) Comparison of the trichoblast cell length (left) and the width (right) in 7 days old seedlings of WT plants with cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi; lines 2.21 and 3.14). Columns are marked with different letters indicating statistically different groups for the trichobalst cell length or with similar letters indicating groups not statistically different for trichoblast cell width according to the ANOVA-and Duncan test ( p

    Journal: PLoS ONE

    Article Title: The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development

    doi: 10.1371/journal.pone.0209407

    Figure Lengend Snippet: CEP2 loss of function results in shortening the cell length and cell width of the longest trichoblasts (double arrows) in the root elongation zone at the level of the transition from the rapid elongation zone to the late elongation zone. The residual CEP2 activity in cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) results in prolonged trichoblasts as compared to cep2 ko mutant plants, but shorter as compared to trichoblasts in WT plants. (A) 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd plants were stained with propidium iodide and analyzed by CLSM (single pictures, 200-fold magnification). Two examples of propidium iodide stained roots are presented for each genotype. (B) Comparison of the trichoblast cell length (left) and the width (right) in 7 days old seedlings of WT plants with cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi; lines 2.21 and 3.14). Columns are marked with different letters indicating statistically different groups for the trichobalst cell length or with similar letters indicating groups not statistically different for trichoblast cell width according to the ANOVA-and Duncan test ( p

    Article Snippet: Propidium iodide and calcofluor-white staining of plant tissues Roots of 7–10 days old seedlings were mounted on a microscope slide, incubated with propidium iodide (20 μg/ml H2 O; Sigma-Aldrich Chemie, Germany) or calcofluor-white (200 μl 500 mM Na-phosphate, 400 μl H2 O, 400 μl calcofluor-white stain pH 7.5; Sigma-Aldrich Chemie, Germany) and examined by CLSM.

    Techniques: Activity Assay, Mutagenesis, Staining, Confocal Laser Scanning Microscopy

    CEP2 loss of function does not impair PCD in the LRC, although the PCD site I is shifted more close to the root tip in cep2 ko mutants due to the reduced cell lengh of the LRC and again to a lesser extent in cep1 cep2 double ko/kd mutants as compared to WT plants. 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) were stained with propidium iodide and analyzed by CLSM (stack of 14 optical sections, 200-fold magnification). PCD site I and II are indicated by nuclei stained with propidium iodide (arrow heads) thus indicating cells approaching PCD since their cell membranes become permeable to propidium iodide.

    Journal: PLoS ONE

    Article Title: The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development

    doi: 10.1371/journal.pone.0209407

    Figure Lengend Snippet: CEP2 loss of function does not impair PCD in the LRC, although the PCD site I is shifted more close to the root tip in cep2 ko mutants due to the reduced cell lengh of the LRC and again to a lesser extent in cep1 cep2 double ko/kd mutants as compared to WT plants. 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) were stained with propidium iodide and analyzed by CLSM (stack of 14 optical sections, 200-fold magnification). PCD site I and II are indicated by nuclei stained with propidium iodide (arrow heads) thus indicating cells approaching PCD since their cell membranes become permeable to propidium iodide.

    Article Snippet: Propidium iodide and calcofluor-white staining of plant tissues Roots of 7–10 days old seedlings were mounted on a microscope slide, incubated with propidium iodide (20 μg/ml H2 O; Sigma-Aldrich Chemie, Germany) or calcofluor-white (200 μl 500 mM Na-phosphate, 400 μl H2 O, 400 μl calcofluor-white stain pH 7.5; Sigma-Aldrich Chemie, Germany) and examined by CLSM.

    Techniques: Staining, Confocal Laser Scanning Microscopy

    CEP2 loss of function results in shortening the longest cells of the lateral root cap (LRC) at the level of the PCD site I. The residual CEP2 activity in cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) results in prolonged LRC cells as compared to cep2 ko mutants, but shorter as compared to LRC cells in WT plants. (A) 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) were stained with propidium iodide and analyzed by CLSM (single pictures, 400-fold magnification). The 3–5 longer most cells of the LRC at the level of the PCD site I (double arrow) are compared. Nuclei stained with propidium iodide are marked (arrow heads) indicating cells approaching PCD since their cell membranes become permeable resulting in propidium iodide stained nuclei in addition to the stained cell wall. (B) Comparison of the length (left) and the width (right) of the 3–5 longer most LRC cells in 7 days old seedlings of WT plants with cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi; lines 2.21 and 3.14) mutants. Columns are marked with different letters indicating statistically different groups for LRC cell length ( p

    Journal: PLoS ONE

    Article Title: The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development

    doi: 10.1371/journal.pone.0209407

    Figure Lengend Snippet: CEP2 loss of function results in shortening the longest cells of the lateral root cap (LRC) at the level of the PCD site I. The residual CEP2 activity in cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) results in prolonged LRC cells as compared to cep2 ko mutants, but shorter as compared to LRC cells in WT plants. (A) 7 days old seedlings of WT, cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi) were stained with propidium iodide and analyzed by CLSM (single pictures, 400-fold magnification). The 3–5 longer most cells of the LRC at the level of the PCD site I (double arrow) are compared. Nuclei stained with propidium iodide are marked (arrow heads) indicating cells approaching PCD since their cell membranes become permeable resulting in propidium iodide stained nuclei in addition to the stained cell wall. (B) Comparison of the length (left) and the width (right) of the 3–5 longer most LRC cells in 7 days old seedlings of WT plants with cep2 ko and cep1 cep2 double ko/kd mutants ( cep1 + cep2- RNAi; lines 2.21 and 3.14) mutants. Columns are marked with different letters indicating statistically different groups for LRC cell length ( p

    Article Snippet: Propidium iodide and calcofluor-white staining of plant tissues Roots of 7–10 days old seedlings were mounted on a microscope slide, incubated with propidium iodide (20 μg/ml H2 O; Sigma-Aldrich Chemie, Germany) or calcofluor-white (200 μl 500 mM Na-phosphate, 400 μl H2 O, 400 μl calcofluor-white stain pH 7.5; Sigma-Aldrich Chemie, Germany) and examined by CLSM.

    Techniques: Activity Assay, Staining, Confocal Laser Scanning Microscopy

    Mean percentage of living/propidium iodide positive fibroblast cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure . For statistical significant relationships, reference Table 3

    Journal: SpringerPlus

    Article Title: The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection

    doi: 10.1186/s40064-016-2757-5

    Figure Lengend Snippet: Mean percentage of living/propidium iodide positive fibroblast cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure . For statistical significant relationships, reference Table 3

    Article Snippet: Propidium iodide Propidium iodide (Sigma-Aldrich) was used as a molecular marker in this experiment because it is typically impermeable to the cell membrane.

    Techniques: Derivative Assay

    Mean percentage of living/propidium iodide positive HeLa cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure . For statistical significant relationships, reference Table 3

    Journal: SpringerPlus

    Article Title: The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection

    doi: 10.1186/s40064-016-2757-5

    Figure Lengend Snippet: Mean percentage of living/propidium iodide positive HeLa cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure . For statistical significant relationships, reference Table 3

    Article Snippet: Propidium iodide Propidium iodide (Sigma-Aldrich) was used as a molecular marker in this experiment because it is typically impermeable to the cell membrane.

    Techniques: Derivative Assay

    RASSF1A induces cell cycle arrest, apoptosis and ANKRD1 expression ( A ) Flow cytometry analysis of YAP1 in TREx293 cells transfected with a GFP control or GFP-RASSF1A plasmid after 72 h treatment with Doxycycline (YAP1 ind.) or without (unind.). Cell cycle distribution was analyzed via flow cytometry using propidium iodid staining ( n = 10 4 cells). ( B ) Nuclear morphology after YAP1 and RASSF1A transfection. mCherry-YAP1 and GFP-RASSF1A and/or control plasmid were transfected in HEK293T cells; nuclei of transfected cells were analyzed by fluorescence microscopy after 72 h ( n = 200 each). ( C ) Quantitative analysis of ANKRD1 expression level in HEK293T cells transfected with control, YAP1 and/or RASSF1A after 72 h. All expression data obtained by qRT-PCR were normalized to GAPDH and Ctrl was set 1. ( D ) Quantitative analysis of ANKRD1 expression level in HeLa cells transfected with control, YAP1 and/or RASSF1A after 72 h. p -values * p

    Journal: Oncotarget

    Article Title: The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth

    doi: 10.18632/oncotarget.18177

    Figure Lengend Snippet: RASSF1A induces cell cycle arrest, apoptosis and ANKRD1 expression ( A ) Flow cytometry analysis of YAP1 in TREx293 cells transfected with a GFP control or GFP-RASSF1A plasmid after 72 h treatment with Doxycycline (YAP1 ind.) or without (unind.). Cell cycle distribution was analyzed via flow cytometry using propidium iodid staining ( n = 10 4 cells). ( B ) Nuclear morphology after YAP1 and RASSF1A transfection. mCherry-YAP1 and GFP-RASSF1A and/or control plasmid were transfected in HEK293T cells; nuclei of transfected cells were analyzed by fluorescence microscopy after 72 h ( n = 200 each). ( C ) Quantitative analysis of ANKRD1 expression level in HEK293T cells transfected with control, YAP1 and/or RASSF1A after 72 h. All expression data obtained by qRT-PCR were normalized to GAPDH and Ctrl was set 1. ( D ) Quantitative analysis of ANKRD1 expression level in HeLa cells transfected with control, YAP1 and/or RASSF1A after 72 h. p -values * p

    Article Snippet: FACS and microarrays Cell cycle distribution was analyzed via flow cytometry (BD FACSCanto) using propidium iodid (Sigma) and pEGFP or pEGFP-RASSF1A transfected TREx293 cells (−/+ doxycycline).

    Techniques: Expressing, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Quantitative RT-PCR

    The inhibition of RIPK1 by Nec-1 decreases surgery-induced necroptosis in D-Gal-induced aged mice. (A) Representative images of propidium iodide (PI) staining (red) in the dentate gyrus. Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). The arrows indicated some typical cells stained by PI. Bar = 50 μm. (B) The PI-positive cells were counted in the DG. (C,D) Western blot analysis of RIP1 (C) and NF-κB (D) in the hippocampus. The upper panel showed the protein bands of RIP1 and NF-κB, and GAPDH was reference control. The lower panel showed that relative integrated density value of RIP1 or NF-KB to GAPDH. Data were expressed as the mean ± SEM. # P

    Journal: Frontiers in Behavioral Neuroscience

    Article Title: Inhibiting RIPK1 Limits Neuroinflammation and Alleviates Postoperative Cognitive Impairments in D-Galactose-Induced Aged Mice

    doi: 10.3389/fnbeh.2018.00138

    Figure Lengend Snippet: The inhibition of RIPK1 by Nec-1 decreases surgery-induced necroptosis in D-Gal-induced aged mice. (A) Representative images of propidium iodide (PI) staining (red) in the dentate gyrus. Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). The arrows indicated some typical cells stained by PI. Bar = 50 μm. (B) The PI-positive cells were counted in the DG. (C,D) Western blot analysis of RIP1 (C) and NF-κB (D) in the hippocampus. The upper panel showed the protein bands of RIP1 and NF-κB, and GAPDH was reference control. The lower panel showed that relative integrated density value of RIP1 or NF-KB to GAPDH. Data were expressed as the mean ± SEM. # P

    Article Snippet: Administration of Propidium Iodide (PI) Propidium iodide (PI; 10 mg/mL, Sigma, St. Louis, MO, USA) labeling was conducted as described previously (Zhang et al., ; Kanno et al., ) to detect plasma permeability, which is a hallmark of necrotic cell death.

    Techniques: Inhibition, Mouse Assay, Staining, Western Blot

    Overexpression of miR-769-3p suppresses proliferation and induces apoptosis of MCF-7 breast tumor cells. (A) MTT assays of MCF-7 cells overexpressing miR-769-3p in normoxia (O 2 ), hypoxia (N 2 ), and reoxygenation (Re-O 2 ). (B) A representative diagram of annexin V binding assay at 24 h of O 2 , N 2 , and Re-O 2 . Left panel: empty vector; right panel: MCF-7 cells overexpressing miR-769-3p. (C) Flow cytometry analysis for apoptosis at 24 h of O 2 , N 2 , and Re-O 2 . using annexin V and propidium iodide in MCF-7 cells overexpressing miR-769-3p. (D) A representative diagram of flow cytometry for analyzing cell cycle at 24 h of reoxygenation. Upper panel: empty vector; lower panel: MCF-7 cells overexpressing miR-769-3p. (E) Quantitative graph of flow cytometry results in panel (D) showing the percentage of cells in each phase of the cell cycle. *, P

    Journal: Scientific Reports

    Article Title: MicroRNA-769-3p Down-regulates NDRG1 and Enhances Apoptosis in MCF-7 Cells During Reoxygenation

    doi: 10.1038/srep05908

    Figure Lengend Snippet: Overexpression of miR-769-3p suppresses proliferation and induces apoptosis of MCF-7 breast tumor cells. (A) MTT assays of MCF-7 cells overexpressing miR-769-3p in normoxia (O 2 ), hypoxia (N 2 ), and reoxygenation (Re-O 2 ). (B) A representative diagram of annexin V binding assay at 24 h of O 2 , N 2 , and Re-O 2 . Left panel: empty vector; right panel: MCF-7 cells overexpressing miR-769-3p. (C) Flow cytometry analysis for apoptosis at 24 h of O 2 , N 2 , and Re-O 2 . using annexin V and propidium iodide in MCF-7 cells overexpressing miR-769-3p. (D) A representative diagram of flow cytometry for analyzing cell cycle at 24 h of reoxygenation. Upper panel: empty vector; lower panel: MCF-7 cells overexpressing miR-769-3p. (E) Quantitative graph of flow cytometry results in panel (D) showing the percentage of cells in each phase of the cell cycle. *, P

    Article Snippet: The DNA contents were evaluated after staining with propidium iodide (PI) solution containing 50 μg/ml propidium iodide (PI) (Sigma), 0.1 mg/ml RNase A (Sigma), 0.05% Triton X-100 (Sigma) in PBS (Life Technologies).

    Techniques: Over Expression, MTT Assay, Binding Assay, Plasmid Preparation, Flow Cytometry, Cytometry

    GDF15 overexpression alters breast cancer cell cycle profile (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: GDF15 overexpression alters breast cancer cell cycle profile (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

    Article Snippet: Fixed cells were incubated in 50 μL propidium iodide buffer (20 μg/mL PI (Sigma), 0.1% Triton-X 100, 200 μg/mL RNaseA (Promega) in DPBS) for 30 minutes in the dark.

    Techniques: Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Clone Assay, Staining, Flow Cytometry, Cytometry

    MYCER stabilization results in accumulation of cells in S/G2 phase and chromatin-bound CDC45 (A) Constitutive knockdown of FBXW7 with MYCER activation results in additive accumulation of cells in S and G2/M phase. MCF10A-MYCER cells with control or FBXW7 knockdown were cultured for 7 days in the absence or presence of 4OHT (MYC OFF or MYC ON). Cells were fixed and stained with propidium iodide for cell cycle analysis. Results are averages of 3 independent experiments and error bars represent the SEM. (B) Inducible knockdown of FBXW7 with MYCER activation results in accumulation of cells in S and G2/M phase. Control or FBXW7 knockdown cells were cultured for seven days with 1μg/ml doxycycline and vehicle or 4OHT every 72 hr. Results are averages of 3 independent experiments and error bars represent the SEM. (C) Loss of FBXW7 with MYCER activation causes accumulation of CDC45 on chromatin. MCF10A-MYCER cells with control or FBXW7 knockdown were cultured for 7 days with or without 4OHT and fractionated. Fraction P3 was analyzed on SDS-PAGE. Shown is a representative image of CDC45 blot, and graph shows the average of 3 independent experiments each normalized to histone H3. Error bars represent the SEM.

    Journal: Oncotarget

    Article Title: MYC is a critical target of FBXW7

    doi:

    Figure Lengend Snippet: MYCER stabilization results in accumulation of cells in S/G2 phase and chromatin-bound CDC45 (A) Constitutive knockdown of FBXW7 with MYCER activation results in additive accumulation of cells in S and G2/M phase. MCF10A-MYCER cells with control or FBXW7 knockdown were cultured for 7 days in the absence or presence of 4OHT (MYC OFF or MYC ON). Cells were fixed and stained with propidium iodide for cell cycle analysis. Results are averages of 3 independent experiments and error bars represent the SEM. (B) Inducible knockdown of FBXW7 with MYCER activation results in accumulation of cells in S and G2/M phase. Control or FBXW7 knockdown cells were cultured for seven days with 1μg/ml doxycycline and vehicle or 4OHT every 72 hr. Results are averages of 3 independent experiments and error bars represent the SEM. (C) Loss of FBXW7 with MYCER activation causes accumulation of CDC45 on chromatin. MCF10A-MYCER cells with control or FBXW7 knockdown were cultured for 7 days with or without 4OHT and fractionated. Fraction P3 was analyzed on SDS-PAGE. Shown is a representative image of CDC45 blot, and graph shows the average of 3 independent experiments each normalized to histone H3. Error bars represent the SEM.

    Article Snippet: To the resulting pellet, RNAse A was added at 1/400, and propidium iodide stock (10mg/ml, Sigma) was added at 1/1000 for flow cytometry.

    Techniques: Activation Assay, Cell Culture, Staining, Cell Cycle Assay, SDS Page

    MCF10A-MYCER cells: a model for MYC deregulation (A) MCF10A-MYCER cells express MYCER protein. Whole cell lysates from uninfected or MYCER-infected MCF10A cells were analyzed by SDS-PAGE. The positions of endogenous MYC and MYCER are indicated. (B) MYCER protein is activated in response to 4OHT treatment. Nuclear translocation of MYCER was analyzed by cell fractionation followed by SDS-PAGE of the chromatin-bound fraction from MCF10A-MYCER cells treated with vehicle or 200nM 4OHT for 48 hr. MYCER increase was normalized to histone H3 levels for quantification. (C) MYCER activation induces accelerated entry into S phase. MCF10A-MYCER cells were arrested at G1/S with double thymidine. Cells were treated with vehicle (ethanol) or 4OHT, then pulse labeled with BrdU 1 hr after release into S phase. BrdU incorporation was assessed using indirect immunofluorescence. PI=propidium iodide was used to stain genomic DNA.

    Journal: Oncotarget

    Article Title: MYC is a critical target of FBXW7

    doi:

    Figure Lengend Snippet: MCF10A-MYCER cells: a model for MYC deregulation (A) MCF10A-MYCER cells express MYCER protein. Whole cell lysates from uninfected or MYCER-infected MCF10A cells were analyzed by SDS-PAGE. The positions of endogenous MYC and MYCER are indicated. (B) MYCER protein is activated in response to 4OHT treatment. Nuclear translocation of MYCER was analyzed by cell fractionation followed by SDS-PAGE of the chromatin-bound fraction from MCF10A-MYCER cells treated with vehicle or 200nM 4OHT for 48 hr. MYCER increase was normalized to histone H3 levels for quantification. (C) MYCER activation induces accelerated entry into S phase. MCF10A-MYCER cells were arrested at G1/S with double thymidine. Cells were treated with vehicle (ethanol) or 4OHT, then pulse labeled with BrdU 1 hr after release into S phase. BrdU incorporation was assessed using indirect immunofluorescence. PI=propidium iodide was used to stain genomic DNA.

    Article Snippet: To the resulting pellet, RNAse A was added at 1/400, and propidium iodide stock (10mg/ml, Sigma) was added at 1/1000 for flow cytometry.

    Techniques: Infection, SDS Page, Translocation Assay, Cell Fractionation, Activation Assay, Labeling, BrdU Incorporation Assay, Immunofluorescence, Staining

    Studies on lens epithelial cells exposed to hyperosmotic solution (500 mOsm). Panel A shows propidium iodide (PI) uptake measured in cells incubated for 30 min in hyperosmotic (500 mOsm) solution (Hyper) or control isosmotic solution (Control) that contained PI (25 µM). The results are expressed as relative fluorescence/mg protein. The values are mean ± SE of results from 6 independent experiments. Panel B shows cytoplasmic calcium concentration measured for 5 min in isosmotic solution (Iso) remained unchanged when hyperosmotic solution (500 mOsm) was introduced for a further 30 min. Data from 15–30 individual cells were averaged and considered as n=1. Results are means ± SE of 5 independent experiments.

    Journal: Experimental eye research

    Article Title: Calcium entry via connexin hemichannels in lens epithelium

    doi: 10.1016/j.exer.2015.01.012

    Figure Lengend Snippet: Studies on lens epithelial cells exposed to hyperosmotic solution (500 mOsm). Panel A shows propidium iodide (PI) uptake measured in cells incubated for 30 min in hyperosmotic (500 mOsm) solution (Hyper) or control isosmotic solution (Control) that contained PI (25 µM). The results are expressed as relative fluorescence/mg protein. The values are mean ± SE of results from 6 independent experiments. Panel B shows cytoplasmic calcium concentration measured for 5 min in isosmotic solution (Iso) remained unchanged when hyperosmotic solution (500 mOsm) was introduced for a further 30 min. Data from 15–30 individual cells were averaged and considered as n=1. Results are means ± SE of 5 independent experiments.

    Article Snippet: Materials Propidium iodide, ethylene glycol tetraacetic acid (EGTA) and DMSO were purchased from Sigma (St Louis, MO, USA).

    Techniques: Incubation, Fluorescence, Concentration Assay

    Studies on lens epithelial cells exposed to hyposmotic solution (200 mOsm). Panel A shows propidium iodide (PI) uptake. Cells were exposed to 25 µM PI for 30 min in hyposmotic (200 mOsm) solution (Hypo) or control isosmotic solution (Con). Some cells were pre-incubated for 60 min in isosmotic solution containing 200 µM GAP27, a connexin inhibitor, before being exposed to either PI/hyposmotic (GAP 27 + Hypo) or PI/isosmotic solution (GAP 27) in the continued presence of GAP 27. Following the PI uptake period, cells were washed, harvested, homogenized, and PI fluorescence intensity was measured. The results are expressed as relative fluorescence/mg protein. The values are mean ± SE of results from 6 independent experiments. *** indicates a significant difference (p

    Journal: Experimental eye research

    Article Title: Calcium entry via connexin hemichannels in lens epithelium

    doi: 10.1016/j.exer.2015.01.012

    Figure Lengend Snippet: Studies on lens epithelial cells exposed to hyposmotic solution (200 mOsm). Panel A shows propidium iodide (PI) uptake. Cells were exposed to 25 µM PI for 30 min in hyposmotic (200 mOsm) solution (Hypo) or control isosmotic solution (Con). Some cells were pre-incubated for 60 min in isosmotic solution containing 200 µM GAP27, a connexin inhibitor, before being exposed to either PI/hyposmotic (GAP 27 + Hypo) or PI/isosmotic solution (GAP 27) in the continued presence of GAP 27. Following the PI uptake period, cells were washed, harvested, homogenized, and PI fluorescence intensity was measured. The results are expressed as relative fluorescence/mg protein. The values are mean ± SE of results from 6 independent experiments. *** indicates a significant difference (p

    Article Snippet: Materials Propidium iodide, ethylene glycol tetraacetic acid (EGTA) and DMSO were purchased from Sigma (St Louis, MO, USA).

    Techniques: Incubation, Fluorescence

    siRNA-mediated targeting of HMGI-C, prolonged G0/G1 arrest in MDA-MB-468 cells. Untreated, (NC) siRNA and 80 pmol HMGI-C treated cells were prepared and stained with the propidium iodide (PI). The percentage of the cell population in untreated,

    Journal: Cell Cycle

    Article Title: HMGI-C suppressing induces P53/caspase9 axis to regulate apoptosis in breast adenocarcinoma cells

    doi: 10.1080/15384101.2016.1190892

    Figure Lengend Snippet: siRNA-mediated targeting of HMGI-C, prolonged G0/G1 arrest in MDA-MB-468 cells. Untreated, (NC) siRNA and 80 pmol HMGI-C treated cells were prepared and stained with the propidium iodide (PI). The percentage of the cell population in untreated,

    Article Snippet: QRT-PCR master mix was purchased from Takara Bio Inc. (Shiga, Japan) and miRNA expression was measured by miRCURY LNA microRNA Reagents (Exiqon), TUNEL and Annexin-V-FLUOS Staining Kit were bought from Roche (Roche, Mannheim, Germany) and propidium iodide dye was purchased from Sigma Aldrich (USA).

    Techniques: Multiple Displacement Amplification, Staining

    Propidium iodide (PI) staining of C . albicans ATCC 90028 exposed to AgNPs for 6 and 24 h. Stained samples were analyzed microscopically. Red fluorescence indicates cells with impaired cell membrane permeability. AgNPs attached to the C . albicans morphotypes are visible as black dots.

    Journal: PLoS ONE

    Article Title: Biogenic nanosilver synthesized in Metarhizium robertsii waste mycelium extract – As a modulator of Candida albicans morphogenesis, membrane lipidome and biofilm

    doi: 10.1371/journal.pone.0194254

    Figure Lengend Snippet: Propidium iodide (PI) staining of C . albicans ATCC 90028 exposed to AgNPs for 6 and 24 h. Stained samples were analyzed microscopically. Red fluorescence indicates cells with impaired cell membrane permeability. AgNPs attached to the C . albicans morphotypes are visible as black dots.

    Article Snippet: The action of AgNPs on the yeast membrane permeability was measured by propidium iodide exclusion (PI, Sigma, USA).

    Techniques: Staining, Fluorescence, Permeability

    Spheroid size. Size measurements of human normal dermal fibroblast (A), colon cancer (B), bladder cancer (C), and metastatic breast cancer (D) spheroids. Left panel: representative fluorescence microscopy images of a spheroid just after electroporation (8 pulses of 100 μs, 1000 V/cm, and 1 Hz) in buffer containing propidium iodide (PI) to visualize electropermeabilized cells and light microscopy images of another spheroid just before treatment (LM). Right panel: Growth curves of the spheroids after treatment with respectively 168 mM calcium (Ca), 1 mM bleomycin (Bleo), electroporation (EP), 168 mM calcium electroporation (Ca EP), electrochemotherapy using 1 mM bleomycin (Bleo EP), and untreated controls (Control). Measurements performed before treatment and at day 2, 3, and 4. Spheroid size is normalized to the size before treatment, means +/- SD, n = 5–6.

    Journal: PLoS ONE

    Article Title: Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

    doi: 10.1371/journal.pone.0144028

    Figure Lengend Snippet: Spheroid size. Size measurements of human normal dermal fibroblast (A), colon cancer (B), bladder cancer (C), and metastatic breast cancer (D) spheroids. Left panel: representative fluorescence microscopy images of a spheroid just after electroporation (8 pulses of 100 μs, 1000 V/cm, and 1 Hz) in buffer containing propidium iodide (PI) to visualize electropermeabilized cells and light microscopy images of another spheroid just before treatment (LM). Right panel: Growth curves of the spheroids after treatment with respectively 168 mM calcium (Ca), 1 mM bleomycin (Bleo), electroporation (EP), 168 mM calcium electroporation (Ca EP), electrochemotherapy using 1 mM bleomycin (Bleo EP), and untreated controls (Control). Measurements performed before treatment and at day 2, 3, and 4. Spheroid size is normalized to the size before treatment, means +/- SD, n = 5–6.

    Article Snippet: Chemicals Propidium iodide (PI; Sigma-Aldrich), bleomycin sulfate (Bleo; Merck-Millipore), and calcium chloride (Ca; SAD, Denmark).

    Techniques: Fluorescence, Microscopy, Electroporation, Light Microscopy

    Palmitoylation of hFasL is required for optimal hFasL processing and cytotoxic activity. ( a ) Diagram of the hFasL transmembrane domain, highlighting the proposed palmitoylation site at aa 82, which we mutated to serine. ( b ) WSU cells stably transfected with constructs encoding for hFasL , hFasLC82S or hFasLA247E were subjected to an acyl-biotinyl exchange protocol as described in Materials and Methods. Specificity of the experiment was controlled by omitting the hydroxylamine (HA) treatment and Fyn was used as an internal control for reaction efficiency. ( c ) JH6.2 cells were co-cultured for the indicated time points with WSU cells stably transfected with hFasL , hFasLC82S or mock. Cell death was then quantified by flow cytometric analysis of the subG1 population of propidium iodide-stained ethanol-fixed cells. The graph represents the average of four independent experiments with error bars indicating the S.D. The level of expression of wild-type hFasL or hFasLC82S at the cell surface of WSU transfected cells was comparable (see the inset). ( d ) Cell lysates of WSU cells stably transfected with constructs encoding for hFasL , hFasLC82S or mock transfected were analyzed by western blotting. ( e ) Levels of soluble FasL were measured in the supernatant of WSU cells stably transfected with wild-type hFasL or hFasLC82S. The graph represents the average of two independent experiments with error bars indicating the S.D.

    Journal: Cell Death & Disease

    Article Title: Palmitoylation of human FasL modulates its cell death-inducing function

    doi: 10.1038/cddis.2010.62

    Figure Lengend Snippet: Palmitoylation of hFasL is required for optimal hFasL processing and cytotoxic activity. ( a ) Diagram of the hFasL transmembrane domain, highlighting the proposed palmitoylation site at aa 82, which we mutated to serine. ( b ) WSU cells stably transfected with constructs encoding for hFasL , hFasLC82S or hFasLA247E were subjected to an acyl-biotinyl exchange protocol as described in Materials and Methods. Specificity of the experiment was controlled by omitting the hydroxylamine (HA) treatment and Fyn was used as an internal control for reaction efficiency. ( c ) JH6.2 cells were co-cultured for the indicated time points with WSU cells stably transfected with hFasL , hFasLC82S or mock. Cell death was then quantified by flow cytometric analysis of the subG1 population of propidium iodide-stained ethanol-fixed cells. The graph represents the average of four independent experiments with error bars indicating the S.D. The level of expression of wild-type hFasL or hFasLC82S at the cell surface of WSU transfected cells was comparable (see the inset). ( d ) Cell lysates of WSU cells stably transfected with constructs encoding for hFasL , hFasLC82S or mock transfected were analyzed by western blotting. ( e ) Levels of soluble FasL were measured in the supernatant of WSU cells stably transfected with wild-type hFasL or hFasLC82S. The graph represents the average of two independent experiments with error bars indicating the S.D.

    Article Snippet: Briefly, cells were fixed with ice-cold 70% ethanol, washed with 38 mM sodium citrate (pH 7.0) and stained for 20 min at 37°C with 69 μ M propidium iodide (Sigma)/38 mM sodium citrate/5 μ g per ml RNaseA (Sigma).

    Techniques: Activity Assay, Stable Transfection, Transfection, Construct, Cell Culture, Flow Cytometry, Staining, Expressing, Western Blot

    Metalloprotease, but not SPPL2a-mediated, FasL processing decreases FasL-induced cell death. WSU cells were pre-treated with inhibitors of either MMP or SPPL2a before co-culture with the Fas-bearing JH6.2 cells (inhibitors remained in media during the co-culture period). Effective inhibition of ADAM10 or SPPL2 was confirmed by western blot. G247 antibody was used to detect the full-length FasL and the antibody Ab-3 was employed for the detection of the N-terminal APL and SPA fragments, ezrin was used as loading control. Cell death was quantified by flow cytometric quantification of the subG1 population of propidium iodide-stained ethanol-fixed cells. ( a ) JH6.2 cells were co-cultured for 6 hours with WSU cells stably transfected with either hFasL or mock transfected, and were either pre-treated with the SPPL2 inhibitor (Z-LL) 2 (2 μ M, 2 h) or left untreated. The graph represents the average of two independent experiments, with error bars indicating the S.D. ( b ) JH6.2 cells were co-cultured for 4 h with WSU cells stably transfected with hFasL or mock transfected and either pre-treated with the MMP inhibitor TAPI-2 (50 μ M, 2 h) or left untreated. The graph represents the average of four independent experiments, with error bars indicating the S.D.

    Journal: Cell Death & Disease

    Article Title: Palmitoylation of human FasL modulates its cell death-inducing function

    doi: 10.1038/cddis.2010.62

    Figure Lengend Snippet: Metalloprotease, but not SPPL2a-mediated, FasL processing decreases FasL-induced cell death. WSU cells were pre-treated with inhibitors of either MMP or SPPL2a before co-culture with the Fas-bearing JH6.2 cells (inhibitors remained in media during the co-culture period). Effective inhibition of ADAM10 or SPPL2 was confirmed by western blot. G247 antibody was used to detect the full-length FasL and the antibody Ab-3 was employed for the detection of the N-terminal APL and SPA fragments, ezrin was used as loading control. Cell death was quantified by flow cytometric quantification of the subG1 population of propidium iodide-stained ethanol-fixed cells. ( a ) JH6.2 cells were co-cultured for 6 hours with WSU cells stably transfected with either hFasL or mock transfected, and were either pre-treated with the SPPL2 inhibitor (Z-LL) 2 (2 μ M, 2 h) or left untreated. The graph represents the average of two independent experiments, with error bars indicating the S.D. ( b ) JH6.2 cells were co-cultured for 4 h with WSU cells stably transfected with hFasL or mock transfected and either pre-treated with the MMP inhibitor TAPI-2 (50 μ M, 2 h) or left untreated. The graph represents the average of four independent experiments, with error bars indicating the S.D.

    Article Snippet: Briefly, cells were fixed with ice-cold 70% ethanol, washed with 38 mM sodium citrate (pH 7.0) and stained for 20 min at 37°C with 69 μ M propidium iodide (Sigma)/38 mM sodium citrate/5 μ g per ml RNaseA (Sigma).

    Techniques: Co-Culture Assay, Inhibition, Western Blot, Flow Cytometry, Staining, Cell Culture, Stable Transfection, Transfection

    Viability staining and morphology of the islets in different culture conditions after the indicated time points detected by confocal laser scanning microscopy. ( a , b columns) monolayer cultures, ( c , d columns) RAFT™ cultures, ( e , f columns) suspension cultures. In order to visualize the integrity of the islets bright field images ( a , c , e columns) were merged with the corresponding fluorescent images ( b , d , f columns). Live cells were visualized by Calcein violet AM ( blue ), apoptotic and dead cells were visualized by Annexin V Alexa Fluor ® 488 ( green ) and propidium-iodide ( red ) staining, (n = 3). All scale bars indicate 100 µm. (Color figure online)

    Journal: Cytotechnology

    Article Title: Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells

    doi: 10.1007/s10616-017-0067-6

    Figure Lengend Snippet: Viability staining and morphology of the islets in different culture conditions after the indicated time points detected by confocal laser scanning microscopy. ( a , b columns) monolayer cultures, ( c , d columns) RAFT™ cultures, ( e , f columns) suspension cultures. In order to visualize the integrity of the islets bright field images ( a , c , e columns) were merged with the corresponding fluorescent images ( b , d , f columns). Live cells were visualized by Calcein violet AM ( blue ), apoptotic and dead cells were visualized by Annexin V Alexa Fluor ® 488 ( green ) and propidium-iodide ( red ) staining, (n = 3). All scale bars indicate 100 µm. (Color figure online)

    Article Snippet: Before the acquisition propidium-iodide (PI, 10 μg/ml, Sigma-Aldrich) was added in AnnexinV binding buffer to dilute AnnexinV Alexa Fluor® 488 5X.

    Techniques: Staining, Confocal Laser Scanning Microscopy

    A. Propidium Iodide uptake - Propidium Iodide (PI) uptake in OHSCs following 35 minute treatment with 3.5mM glutamate. PI staining indicates increased cell death in glutamate treated slices as compared to controls at both 24 and 72 hours following treatment.

    Journal: Brain research

    Article Title: An organotypic hippocampal slice culture model of excitotoxic injury induced spontaneous recurrent epileptiform discharges

    doi: 10.1016/j.brainres.2010.11.065

    Figure Lengend Snippet: A. Propidium Iodide uptake - Propidium Iodide (PI) uptake in OHSCs following 35 minute treatment with 3.5mM glutamate. PI staining indicates increased cell death in glutamate treated slices as compared to controls at both 24 and 72 hours following treatment.

    Article Snippet: Delayed neuronal death was assessed 24 and 72 hours after glutamate treatment by measuring uptake of the fluorescent dye Propidium Iodide (PI, Sigma), using established procedures ( ; ).

    Techniques: Staining

    Bile acid-induced cell injury is dependent on CD38. Acinar cells were pretreated with increasing concentrations of nicotinamide for 30 min prior to a 4-h incubation with TLCS (500 μ m ). Cell injury was measured by percent LDH release ( A ) and propidium

    Journal: The Journal of Biological Chemistry

    Article Title: Cluster of Differentiation 38 (CD38) Mediates Bile Acid-induced Acinar Cell Injury and Pancreatitis through Cyclic ADP-ribose and Intracellular Calcium Release *

    doi: 10.1074/jbc.M113.494534

    Figure Lengend Snippet: Bile acid-induced cell injury is dependent on CD38. Acinar cells were pretreated with increasing concentrations of nicotinamide for 30 min prior to a 4-h incubation with TLCS (500 μ m ). Cell injury was measured by percent LDH release ( A ) and propidium

    Article Snippet: For propidium iodide uptake, acinar cells were incubated in a 48-well plate with 50 μg/ml propidium iodide (Sigma) for 30 min prior to addition of 500 μ m TLCS.

    Techniques: Incubation

    Cytotoxic effect of Pb(NO 3 ) 2 to HL-60 cells. HL-60 cells were cultured in the absence or presence of Pb(NO 3 ) 2 for 24 h. Cell viability was determined based on the propidium iodide assay. Each point represents a mean value of 3 experiments with 6 replicates per concentration. p

    Journal: International Journal of Environmental Research and Public Health

    Article Title: DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    doi: 10.3390/ijerph13010056

    Figure Lengend Snippet: Cytotoxic effect of Pb(NO 3 ) 2 to HL-60 cells. HL-60 cells were cultured in the absence or presence of Pb(NO 3 ) 2 for 24 h. Cell viability was determined based on the propidium iodide assay. Each point represents a mean value of 3 experiments with 6 replicates per concentration. p

    Article Snippet: Fetal bovine serum (FBS), phosphate buffered saline (PBS), and propidium assay were obtained from Sigma Chemical Company (St. Louis, MO, USA).

    Techniques: Cell Culture, Concentration Assay

    Lead nitrate-induced cell cycle arrest in human leukemia (HL-60) cells. Cells were cultured for 24 h with different concentrations of Pb(NO 3 ) 2 . Cell cycle distribution was measured by the propidium iodide staining method. Representative results from at least 3 different experiments are shown.

    Journal: International Journal of Environmental Research and Public Health

    Article Title: DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    doi: 10.3390/ijerph13010056

    Figure Lengend Snippet: Lead nitrate-induced cell cycle arrest in human leukemia (HL-60) cells. Cells were cultured for 24 h with different concentrations of Pb(NO 3 ) 2 . Cell cycle distribution was measured by the propidium iodide staining method. Representative results from at least 3 different experiments are shown.

    Article Snippet: Fetal bovine serum (FBS), phosphate buffered saline (PBS), and propidium assay were obtained from Sigma Chemical Company (St. Louis, MO, USA).

    Techniques: Cell Culture, Staining

    Representative dot plots and histograms showing cell cycle distribution in lead nitrate-treated HL-60 cells. Cell cycle distribution was determined by the propidium iodide staining method, and stained cells were analyzed by flow cytometry (FACS Calibar; Becton-Dickinson) using CellQuest software. A total of 10,000 cells were analyzed per sample. ( A ) control; ( B ) 10 μg/mL Pb(NO 3 ) 2 ; ( C ) 20 μg/mL Pb(NO 3 ) 2 ; ( D ) and 30 μg/mL Pb(NO 3 ) 2 . Three experiments were performed, and 1 representative experiment is shown.

    Journal: International Journal of Environmental Research and Public Health

    Article Title: DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    doi: 10.3390/ijerph13010056

    Figure Lengend Snippet: Representative dot plots and histograms showing cell cycle distribution in lead nitrate-treated HL-60 cells. Cell cycle distribution was determined by the propidium iodide staining method, and stained cells were analyzed by flow cytometry (FACS Calibar; Becton-Dickinson) using CellQuest software. A total of 10,000 cells were analyzed per sample. ( A ) control; ( B ) 10 μg/mL Pb(NO 3 ) 2 ; ( C ) 20 μg/mL Pb(NO 3 ) 2 ; ( D ) and 30 μg/mL Pb(NO 3 ) 2 . Three experiments were performed, and 1 representative experiment is shown.

    Article Snippet: Fetal bovine serum (FBS), phosphate buffered saline (PBS), and propidium assay were obtained from Sigma Chemical Company (St. Louis, MO, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, FACS, Software

    Capacity of DHCA to decrease apoptosis features induced by UVB. L929 fibroblasts were pretreated with 35 μ M DHCA or 1 mM NAC for 1 h, irradiated with UVB (600 mJ/cm 2 ), and after 24 h, analyses were performed. (a) Cells were stained with annexin V, and apoptotic cells were quantified by flow cytometry. (b) Cells were double stained with acridine orange and propidium iodide, and viable cells (green fluorescence nuclei), late apoptotic cells (orange fluorescence nuclei, white headless arrow), and necrotic cells (red fluorescence nuclei, white arrows) were observed on fluorescence microscopy and quantitated. (c) Active caspase 9 expression was determined and quantified by western blot analysis using specific antibodies. (d) Cells were stained with Hoechst 33342 stain, and cells with condensed nuclei (white arrows) were observed on fluorescence microscopy and quantitated. Each column represents the mean ± SD ( n = 3). ### p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Dihydrocaffeic Acid Prevents UVB-Induced Oxidative Stress Leading to the Inhibition of Apoptosis and MMP-1 Expression via p38 Signaling Pathway

    doi: 10.1155/2019/2419096

    Figure Lengend Snippet: Capacity of DHCA to decrease apoptosis features induced by UVB. L929 fibroblasts were pretreated with 35 μ M DHCA or 1 mM NAC for 1 h, irradiated with UVB (600 mJ/cm 2 ), and after 24 h, analyses were performed. (a) Cells were stained with annexin V, and apoptotic cells were quantified by flow cytometry. (b) Cells were double stained with acridine orange and propidium iodide, and viable cells (green fluorescence nuclei), late apoptotic cells (orange fluorescence nuclei, white headless arrow), and necrotic cells (red fluorescence nuclei, white arrows) were observed on fluorescence microscopy and quantitated. (c) Active caspase 9 expression was determined and quantified by western blot analysis using specific antibodies. (d) Cells were stained with Hoechst 33342 stain, and cells with condensed nuclei (white arrows) were observed on fluorescence microscopy and quantitated. Each column represents the mean ± SD ( n = 3). ### p

    Article Snippet: Acridine Orange and Propidium Iodide Double Staining To distinguish viable, late apoptotic, and necrotic cells, double staining with acridine orange (Sigma-Aldrich, St. Louis, MO, USA) and propidium iodide (Invitrogen, Eugene, OR, USA) was performed [ ].

    Techniques: Irradiation, Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Expressing, Western Blot

    Nestin-positive MSC conditioned medium act on the GFAP-positive cell death but not on cell proliferation. (A) Characterization of committed cell types in proliferating neurospheres in presence of EGF. We observed that 14.4 ± 7.1%, 7.1± 4.1% and 3.4 ± 1.6% of cells were respectively GFAP-, Tuj1- and O4-positive (these data were obtained by absolute counts on 1576 cells). (B-C) The proliferative capacity of the differentiating neurosphere-derived cells placed in npMSC conditioned medium, nnMSC conditioned medium and in control medium (DEM/F12 + B27) was compared. BrdU incorporation was performed after 48 hours and 4 days in differentiating conditions. Double labelling GFAP-, Tuj1- and O4 with BrdU were performed. The BrdU incorporation by differentiating neurosphere-derived cells placed in the various conditions did not shown significant differences (Statistical test ANOVA, P > 0.05). (D-E) The cell death quantified by propidium iodide incorporation and counting was analysed after 48 hours and 4 days in differentiating conditions and in GFAP-, Tuj1- and O4-positive cells. After 48 hours, a significant decrease of the number of GFAP-positive cells which have incorporated the propidium iodide is observed (***Student T test, p

    Journal: BMC Neuroscience

    Article Title: Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4

    doi: 10.1186/1471-2202-5-33

    Figure Lengend Snippet: Nestin-positive MSC conditioned medium act on the GFAP-positive cell death but not on cell proliferation. (A) Characterization of committed cell types in proliferating neurospheres in presence of EGF. We observed that 14.4 ± 7.1%, 7.1± 4.1% and 3.4 ± 1.6% of cells were respectively GFAP-, Tuj1- and O4-positive (these data were obtained by absolute counts on 1576 cells). (B-C) The proliferative capacity of the differentiating neurosphere-derived cells placed in npMSC conditioned medium, nnMSC conditioned medium and in control medium (DEM/F12 + B27) was compared. BrdU incorporation was performed after 48 hours and 4 days in differentiating conditions. Double labelling GFAP-, Tuj1- and O4 with BrdU were performed. The BrdU incorporation by differentiating neurosphere-derived cells placed in the various conditions did not shown significant differences (Statistical test ANOVA, P > 0.05). (D-E) The cell death quantified by propidium iodide incorporation and counting was analysed after 48 hours and 4 days in differentiating conditions and in GFAP-, Tuj1- and O4-positive cells. After 48 hours, a significant decrease of the number of GFAP-positive cells which have incorporated the propidium iodide is observed (***Student T test, p

    Article Snippet: BrdU and propidium iodide incorporation After 24 hours or 3 days of culture, BrdU (20 μM, Sigma) which is a S-phase marker, or propidium iodide (400 mg/ml, Sigma) was added to the differentiating neurosphere cultures for 18 hours before fixation and staining.

    Techniques: Activated Clotting Time Assay, Derivative Assay, BrdU Incorporation Assay

    Analysis of apoptosis in the human MG-63 osteosarcoma cell line following treatment with resveratrol alone or in combination with the GSK3β inhibitor, chir99021. Following drug treatment for 72 h, the cells were labeled with annexin V and PI and the apoptosis was detected using flow cytometry. (A) 0 µ g/ml, (B) 10 µ g/ml, (C) 20 µ g/ml and (D) 40 µ g/ml resveratrol, (E) dimethyl sulfoxide, (F) 3 µ M chir99021+10 µ g/ml, (G) 3 µ M chir99021+20 µ g/ml, (H) 3 µ M chir99021+40 µ g/ml resveratrol. Upper left quadrant, apoptotic cells; upper right, dead and late apoptotic cells; lower left, living cells; lower right, mechanically injured cells. PI, propidium iodide.

    Journal: Molecular Medicine Reports

    Article Title: Resveratrol inhibits canonical Wnt signaling in human MG-63 osteosarcoma cells

    doi: 10.3892/mmr.2015.4338

    Figure Lengend Snippet: Analysis of apoptosis in the human MG-63 osteosarcoma cell line following treatment with resveratrol alone or in combination with the GSK3β inhibitor, chir99021. Following drug treatment for 72 h, the cells were labeled with annexin V and PI and the apoptosis was detected using flow cytometry. (A) 0 µ g/ml, (B) 10 µ g/ml, (C) 20 µ g/ml and (D) 40 µ g/ml resveratrol, (E) dimethyl sulfoxide, (F) 3 µ M chir99021+10 µ g/ml, (G) 3 µ M chir99021+20 µ g/ml, (H) 3 µ M chir99021+40 µ g/ml resveratrol. Upper left quadrant, apoptotic cells; upper right, dead and late apoptotic cells; lower left, living cells; lower right, mechanically injured cells. PI, propidium iodide.

    Article Snippet: Subsequently, the suspension was washed with PBS and resuspended in 190 µ l binding buffer, 10 µ l propidium iodide (PI; Sigma-Aldrich) was added.

    Techniques: Labeling, Flow Cytometry, Cytometry

    Haptotaxis assays of MDA-MB-435 metastatic variants. Each variant was adjusted to 500,000 cells and applied to Transwell chambers in a haptotaxis assay, where the underside of the membrane was coated with matrigel. Cells migrating to the underside of the chamber were stained with propidium iodide and counted under 400× Magnification. Bars represent ±SEM. Data are representative of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments ( P

    Journal: Breast Cancer Research

    Article Title: Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells

    doi: 10.1186/bcr1329

    Figure Lengend Snippet: Haptotaxis assays of MDA-MB-435 metastatic variants. Each variant was adjusted to 500,000 cells and applied to Transwell chambers in a haptotaxis assay, where the underside of the membrane was coated with matrigel. Cells migrating to the underside of the chamber were stained with propidium iodide and counted under 400× Magnification. Bars represent ±SEM. Data are representative of three independent experiments. Treatments denoted by the same letter indicate no significant difference between those treatments. Treatments denoted by different letters indicate a significant difference between those treatments ( P

    Article Snippet: Abbreviations Arp2/3 = actin related protein 2/3; BSA = bovine serum albumin; FBS = fetal bovine serum; FITC = fluorescein isothyocianate; PBS = phosphate-buffered saline; PI = propidium iodide; WASP = Wiskott Aldrich Syndrome protein.

    Techniques: Multiple Displacement Amplification, Variant Assay, Staining

    Inhibition of Rho GTPases, Rac, or Cdc42 in migration of the high metastatic MDA-MB-435α6HG6 cell variant. (a) MDA-MB-435α6HG6 cells were treated with vehicle or 2 ng/ml toxin B for 24 h and subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. (b) MDA-MB-435α6HG6 cells transiently expressing vector alone or myc-Cdc42(T17N) were subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. Equal loading was confirmed by a total actin blot, ectopic myc-Cdc42(T17N) expression confirmed by western blotting with anti-Cdc42 or anti-myc. (c) MDA-MB-435α6HG6 cells transiently expressing vector alone, myc-Rac1(T17N), or myc-Rac3(T17N) were subjected to a basement membrane haptotaxis assay. Bars represent ±SEM; equal loading was confirmed by total actin blot. Myc-Rac1(T17N) and myc-Rac3(T17N) expression were confirmed by western blotting with anti-Rac or anti-myc. Data are expressed as mean ±SEM of three independent experiments. A star denotes statistical significance from control ( P

    Journal: Breast Cancer Research

    Article Title: Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells

    doi: 10.1186/bcr1329

    Figure Lengend Snippet: Inhibition of Rho GTPases, Rac, or Cdc42 in migration of the high metastatic MDA-MB-435α6HG6 cell variant. (a) MDA-MB-435α6HG6 cells were treated with vehicle or 2 ng/ml toxin B for 24 h and subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. (b) MDA-MB-435α6HG6 cells transiently expressing vector alone or myc-Cdc42(T17N) were subjected to a basement membrane haptotaxis assay. Cells migrating to the underside of the membrane were stained with propidium iodide and counted under 400× Magnification. Equal loading was confirmed by a total actin blot, ectopic myc-Cdc42(T17N) expression confirmed by western blotting with anti-Cdc42 or anti-myc. (c) MDA-MB-435α6HG6 cells transiently expressing vector alone, myc-Rac1(T17N), or myc-Rac3(T17N) were subjected to a basement membrane haptotaxis assay. Bars represent ±SEM; equal loading was confirmed by total actin blot. Myc-Rac1(T17N) and myc-Rac3(T17N) expression were confirmed by western blotting with anti-Rac or anti-myc. Data are expressed as mean ±SEM of three independent experiments. A star denotes statistical significance from control ( P

    Article Snippet: Abbreviations Arp2/3 = actin related protein 2/3; BSA = bovine serum albumin; FBS = fetal bovine serum; FITC = fluorescein isothyocianate; PBS = phosphate-buffered saline; PI = propidium iodide; WASP = Wiskott Aldrich Syndrome protein.

    Techniques: Inhibition, Migration, Multiple Displacement Amplification, Variant Assay, Staining, Expressing, Plasmid Preparation, Western Blot

    Fluorescence micrograph of propidium iodide (PI) cellular uptake in MCF-7 cells by electroporation using 50 pulses and frequency 1 Hz in the absence ( a ) and presence ( b ) of multi-walled carbon nanotubes (MWCNTs) (30 μg/mL). The field intensity of main pulses (E M ) was 20 V/cm and the alignment field (E A ) intensity was 15 V/cm. Cell suspension samples in the same density (10 6 cells/mL) were treated as described above and stained with PI and viewed under microscope as described in the Experimental Section . As cell density was identical in both samples, only fluorescence images were shown here.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumour Cell Membrane Poration and Ablation by Pulsed Low-Intensity Electric Field with Carbon Nanotubes

    doi: 10.3390/ijms16046890

    Figure Lengend Snippet: Fluorescence micrograph of propidium iodide (PI) cellular uptake in MCF-7 cells by electroporation using 50 pulses and frequency 1 Hz in the absence ( a ) and presence ( b ) of multi-walled carbon nanotubes (MWCNTs) (30 μg/mL). The field intensity of main pulses (E M ) was 20 V/cm and the alignment field (E A ) intensity was 15 V/cm. Cell suspension samples in the same density (10 6 cells/mL) were treated as described above and stained with PI and viewed under microscope as described in the Experimental Section . As cell density was identical in both samples, only fluorescence images were shown here.

    Article Snippet: 400 μL cell suspension mixed with 80 μL Trypan Blue (TB; 0.4%, v /w ) or 2 μL propidium iodide (PI, Abcam, Cambridge, UK; Cat# ab14085) was placed in the electroporation cuvette.

    Techniques: Fluorescence, Electroporation, Staining, Microscopy

    Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Journal: Bioengineering

    Article Title: GSK461364A, a Polo-Like Kinase-1 Inhibitor Encapsulated in Polymeric Nanoparticles for the Treatment of Glioblastoma Multiforme (GBM)

    doi: 10.3390/bioengineering5040083

    Figure Lengend Snippet: Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Article Snippet: Propidium iodide (PI), Annexin binding buffer and phosphate buffer saline (PBS) were purchased from abcam (Cambridge, MA, USA).

    Techniques: Staining

    The effect of iASPP 764–780 on staurosporine (STS)-induced apoptosis and viability of MDA-MB-231 cancer cell spheroids. Apoptosis in spheroids was measured using Annexin V FITC in a time dependent manner. (A) Upper panel: representative images show green fluorescence of spheroids undergoing apoptosis following 24 h. (a) Untreated cells, (b) cells treated with iASPP 764–780 without STS, (c) cells treated with STS without iASPP 764–780, (d) cells treated with iASPP 764–780 and STS. Lower panel: time-dependent apoptosis in spheroids of MDA-MB-231 cancer cells: the graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 abolished apoptosis induced by STS. (B) Time-dependent inhibition of STS-induced cytotoxicity by iASPP 764–780 in the spheroids of MDA-MB-231 cancer cells. Cytotoxicity was measured using propidium iodide (P.I). The graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 inhibited the cytotoxicity of STS by about 40%. (C) IC 50 value of iASPP 764–780 in inhibiting apoptosis in MDA-MB-231 cells. The IC 50 value was found to be 13 ± 1 μM with maximum inhibition of 80%. The EC 50 was found to be 7.5 ± 0.6 μM. (D) Time-dependent inhibition of STS-induced cytotoxicity by iASPP 764–780 in spheroids of PC-3 cancer cells. The graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 inhibited the cytotoxicity of STS by about 50%. (E) The effect of iASPP 764–780 on the viability of MDA-MB-231 cancer cell spheroids with different levels of NAF-1 expression/function. Four different cell lines were used: WT, NAF-1(–), NAF-1(+), and H114C. The percentage of cell death inhibition induced by iASPP 764–780 was obtained by dividing the fluorescence intensity of P.I, indicative of cell death, by the control fluorescence intensity of PI in the absence of iASPP 764–780. The results are shown as average ± SD of four independent experiments. iASPP 764–780 had the most significant effect on cell death inhibition in H114C cells and the less significant effect on cell death inhibition in NAF-1(–) cells. ** p ≤ 0.01.

    Journal: Chemical Science

    Article Title: The anti-apoptotic proteins NAF-1 and iASPP interact to drive apoptosis in cancer cells anti-apoptotic proteins NAF-1 and iASPP interact to drive apoptosis in cancer cells †Electronic supplementary information (ESI) available: Experimental section Fig. S1–S4 and Tables S1–S4. See DOI: 10.1039/c8sc03390k

    doi: 10.1039/c8sc03390k

    Figure Lengend Snippet: The effect of iASPP 764–780 on staurosporine (STS)-induced apoptosis and viability of MDA-MB-231 cancer cell spheroids. Apoptosis in spheroids was measured using Annexin V FITC in a time dependent manner. (A) Upper panel: representative images show green fluorescence of spheroids undergoing apoptosis following 24 h. (a) Untreated cells, (b) cells treated with iASPP 764–780 without STS, (c) cells treated with STS without iASPP 764–780, (d) cells treated with iASPP 764–780 and STS. Lower panel: time-dependent apoptosis in spheroids of MDA-MB-231 cancer cells: the graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 abolished apoptosis induced by STS. (B) Time-dependent inhibition of STS-induced cytotoxicity by iASPP 764–780 in the spheroids of MDA-MB-231 cancer cells. Cytotoxicity was measured using propidium iodide (P.I). The graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 inhibited the cytotoxicity of STS by about 40%. (C) IC 50 value of iASPP 764–780 in inhibiting apoptosis in MDA-MB-231 cells. The IC 50 value was found to be 13 ± 1 μM with maximum inhibition of 80%. The EC 50 was found to be 7.5 ± 0.6 μM. (D) Time-dependent inhibition of STS-induced cytotoxicity by iASPP 764–780 in spheroids of PC-3 cancer cells. The graphs show untreated cells (blue), cells treated with STS (red), cells treated with iASPP 764–780 (green), and cells treated with STS and iASPP 764–780 (violet). iASPP 764–780 inhibited the cytotoxicity of STS by about 50%. (E) The effect of iASPP 764–780 on the viability of MDA-MB-231 cancer cell spheroids with different levels of NAF-1 expression/function. Four different cell lines were used: WT, NAF-1(–), NAF-1(+), and H114C. The percentage of cell death inhibition induced by iASPP 764–780 was obtained by dividing the fluorescence intensity of P.I, indicative of cell death, by the control fluorescence intensity of PI in the absence of iASPP 764–780. The results are shown as average ± SD of four independent experiments. iASPP 764–780 had the most significant effect on cell death inhibition in H114C cells and the less significant effect on cell death inhibition in NAF-1(–) cells. ** p ≤ 0.01.

    Article Snippet: The resulting spheroids were then treated with STS and the annexin V FITC reagent (abcam) to detect apoptosis, or propidium iodide reagent (abcam) to detect cell death.

    Techniques: Multiple Displacement Amplification, Fluorescence, Inhibition, Expressing

    SU11652 induces apoptosis of MV - 4 - 11 cells. MV-4-11 cells were incubated with 0, 10 and 100 nM SU11652 for 24 hours. Cells were stained with Cy5-labeled annexin V and propidium iodide, followed by analyses with a flow cytometry. Percentages of annexin V-positive and propidium iodide-negative cells, that is, apoptotic cells, are indicated.

    Journal: Journal of Hematology & Oncology

    Article Title: SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells

    doi: 10.1186/1756-8722-5-72

    Figure Lengend Snippet: SU11652 induces apoptosis of MV - 4 - 11 cells. MV-4-11 cells were incubated with 0, 10 and 100 nM SU11652 for 24 hours. Cells were stained with Cy5-labeled annexin V and propidium iodide, followed by analyses with a flow cytometry. Percentages of annexin V-positive and propidium iodide-negative cells, that is, apoptotic cells, are indicated.

    Article Snippet: To assess cell cycle arrest, the cells were fixed with ethanol overnight and then stained with propidium iodide in the presence of RNAse.

    Techniques: Incubation, Staining, Labeling, Flow Cytometry, Cytometry

    SU11652 induces cell cycle arrest of MV - 4 - 11 cells. MV-4-11 cells were incubated with 0, 10, and 100 nM SU11652 for 24 hours. Cells were fixed with ethanol and stained with propidium iodide before flow cytometric analysis. Percentages of cells in G1, S, and G2 phases were calculated by using the ModFit software.

    Journal: Journal of Hematology & Oncology

    Article Title: SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells

    doi: 10.1186/1756-8722-5-72

    Figure Lengend Snippet: SU11652 induces cell cycle arrest of MV - 4 - 11 cells. MV-4-11 cells were incubated with 0, 10, and 100 nM SU11652 for 24 hours. Cells were fixed with ethanol and stained with propidium iodide before flow cytometric analysis. Percentages of cells in G1, S, and G2 phases were calculated by using the ModFit software.

    Article Snippet: To assess cell cycle arrest, the cells were fixed with ethanol overnight and then stained with propidium iodide in the presence of RNAse.

    Techniques: Incubation, Staining, Flow Cytometry, Software

    Effect of BAG4 on the cell cycle and apoptosis of GC cells. Representative histograms depicting cell cycle profiles of (A) BAG4-knockdown SGC7901 and (B) BAG4-overexpressing AGS cells. The percentage of apoptotic (C) SGC7901 and (D) AGS cells. BAG4, Bcl-2, associated athanogene 4; sh, short hairpin; NC, negative control; PI, propidium iodide.

    Journal: Molecular Medicine Reports

    Article Title: Bcl-2 associated athanogene 4 promotes proliferation, migration and invasion of gastric cancer cells

    doi: 10.3892/mmr.2017.7073

    Figure Lengend Snippet: Effect of BAG4 on the cell cycle and apoptosis of GC cells. Representative histograms depicting cell cycle profiles of (A) BAG4-knockdown SGC7901 and (B) BAG4-overexpressing AGS cells. The percentage of apoptotic (C) SGC7901 and (D) AGS cells. BAG4, Bcl-2, associated athanogene 4; sh, short hairpin; NC, negative control; PI, propidium iodide.

    Article Snippet: Cells were subsequently harvested and stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), according to the manufacturer's protocol (BioVision, Inc., Milpitas, CA, USA).

    Techniques: Negative Control