propidium iodide Search Results


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  • 99
    Thermo Fisher propidium iodide 1 0 solution in water
    Propidium Iodide 1 0 Solution In Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore propidium iodide
    ATR signaling is activated by A3A expression. (A) A3A activates DNA damage checkpoints. Inducible cell lines were treated with doxycycline for 24 hours and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk2 (T68), phosphorylated Chk1 (S345 and S317), phosphorylated ATM (S1981), Chk2, Chk1, ATM, and ATR. GAPDH was used as a loading control. (B) A3A expression results in cell cycle arrest. Inducible cells lines were treated with doxycycline for 24 or 36 hours, then pulsed with BrdU for 40 minutes prior to harvest. Cells were fixed, stained with anti-BrdU antibody and 7-AAD, and analyzed by flow cytometry. The percentage of cells in each gate are indicated. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 36 hours post-induction. (C) A3A activates ATR signaling in ATM-deficient cells. Inducible AT22IJE-A3A cells with mutant ATM or wild-type ATM were treated with doxycycline to induce A3A expression and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk1 (S345 and S317), Chk1, phosphorylated ATM (S1981), ATM, and ATR. (D) Cell-cycle progression following A3A induction in AT22IJE cells with mutant ATM or wild-type ATM was determined by <t>propidium</t> iodide staining. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 24 hours after treatment, which was similar to results obtained 48 hours after treatment. Results are representative of 3 independent experiments.
    Propidium Iodide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 70351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson propidium iodide
    DAPK1 Regulates Apoptosis in Lymphoid Cells (A) DAPK1 expression in Jurkat cells stably transfected with either vector alone, DAPK1 siRNA-A, or DAPK1 siRNA-C as measured by western blot. Tubulin expression served as a control. (B) Percent live cells were measured in Jurkat cells stably transfected with vector alone or DAPK1 siRNA-C, treated with activating-Fas antibody (100 ng/ml). After 16 hr, cells were harvested and suspended in binding buffer with annexin V-FITC and <t>propidium</t> iodide, followed by flow cytometry to assess cell death. Error bars indicate SD. (C) p53 expression in cells treated with either no, 50 ng/ml, or 100 ng/ml Fas-activating antibody.
    Propidium Iodide, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 33902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher propidium iodide
    Cx43 expression and localization during fibroblast to myofibroblast transition and related PRP effects. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. The cells cultured in proliferation medium (PM) were used as control undifferentiated cells. ( A ) RT-PCR analysis of Cx43 expression in the indicated experimental conditions. Representative agarose gel is shown. The densitometric analysis of the bands normalized to β-actin is reported in the histogram. ( B ) Western Blotting analysis of Cx43 expression. Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( C – E ) Representative superimposed differential interference contrast (DIC, grey) and confocal fluorescence images of the cells immunostained with antibodies against Cx43 (green) and counterstained with <t>propidium</t> iodide (PI, red) to label nuclei. Scale bar: 30 µm. Scale bar in the inset in D: 15 µm. Arrows indicate the localization of Cx43 at the membrane level of two adjacent cells. ( F ) Histogram showing the densitometric analysis of the intensity of the Cx43 fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (12). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p
    Propidium Iodide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 42022 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson propidium iodide pi
    HsLin06_18-FITC uptake and membrane permeabilization in Candida albicans biofilms treated with the [caspofungin + HsLin06_18-FITC] combination. Confocal microscope images of 24 h treated C. albicans biofilms with 0.625 μM caspofungin (CASPO) and/or 0.5 μM HsLin06_18-FITC and 30 μg/mL <t>propidium</t> iode (PI). Bar: 5 μm.
    Propidium Iodide Pi, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 15335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher propidium iodide pi
    Naegleria fowleri trophozoites incubated with IC 90 of STS for 24 h ( D – F ). Negative control ( A – C ). Hoechst stain is different in control cells, where no fluorescence is observed ( B ), and in treated cells, where the nuclei are intense blue ( E ). Red fluorescence corresponds to the <t>propidium</t> iodide stain ( C , F ). Visible channel ( A , D ); Hoechst channel ( B , E ), and propidium iodide channel ( C , F ). Images (40×) are representative of the cell population observed in the performed experiments. Images were obtained using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. Obtained results were similar for both N. fowleri type strains.
    Propidium Iodide Pi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson propidium iodide staining
    RUNX1 silencing deregulates breast cancer cell mitosis. RUNX1 was knocked down in MCF7 cells either constitutively ( a (left) and b ) or conditionally upon treatment with dox ( a (right), c , d g and h ). Cells were maintained in medium supplemented with either complete serum ( a , c , d , f – h ) or CSS ( b ). ( a , b ) Cell cycle profiles were obtained by FACS analysis of <t>propidium</t> iodide-stained cells. In b , cells were treated with either vehicle control (EtOH) or estradiol (E2) for 48 h as indicated. * P
    Propidium Iodide Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime propidium iodide
    miR-18a induces DNA damage. a Expression of γ-H2AX as measured by immunofluorescence in Raji cells; EBV-positive Raji cells were stained with anti-γ-H2AX and DAPI. Expression of γ-H2AX is indicated as green loci. DAPI was used to stain the cell nuclei. The merge images present the DAPI and FITC as blue and green, respectively. b Expression of γ-H2AX as measured by western blotting in EBV-positive or -negative cells. c Detection of DNA damage after transfection of miR-18a. The comet assay was applied. Cells were electrophoresed in agarose gels on a coverslip and were stained with <t>propidium</t> iodide. Labeled DNA was visualized under a fluorescence microscope. d Detection of DNA damage after transfection of miR-18a in EBV-negative and -positive BJAB cells. Magnification, × 100. e Graphic presentation of all chromosomal changes. Cells transfected with miR-18a and mimics negative control were analyzed by comparative genomic hybridization array (Array-CGH). The regions of DNA gain (blue) and loss (red) are shown
    Propidium Iodide, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 1778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Keygen Biotech propidium iodide
    BaF3/ITD-R and MV4-11-R cells are highly resistant to sorafenib. a BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with the indicated concentrations of sorafenib for 48 h and subjected to an Annexin-V/PI assay. The percentage values indicate the total cell death populations. b BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with various concentrations of sorafenib for 72 h and subjected to an MTS assay. The numbers on the curves indicate the IC 50 of sorafenib in each cell line. PI <t>propidium</t> iodide
    Propidium Iodide, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 94/100, based on 1347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Zeiss propidium iodide
    Effect of Ia on P. aeruginosa biofilms. ( a ) Untreated GFP-labelled PA14 biofilm; ( b ) GFP- labelled PA14 biofilm grown with Ia at 8 µM; ( c ) GFP-labelled PA14 biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( d ) GFP-labelled PA14 biofilm grown with 8 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( e ) Biomass quantitation of PA14 biofilms; ( f ) Untreated GFP-labelled PAO1-L biofilm; ( g ) GFP-labelled PAO1-L biofilm grown with Ia at 34 µM; ( h ) GFP-labelled PAO1-L biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( i ) GFP-labelled PAO1-L biofilm grown with 34 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of incubation; ( j ) Biomass quantitation of PAO1-L biofilms. Dead cells and extracellular DNA were stained with <t>propidium</t> iodide (PI). Three-dimensional (3D) sections and cross sections are shown. Scale bars represent 100 µm.
    Propidium Iodide, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 92/100, based on 1404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime propidium iodide pi
    Cell cycle analysis of A549 cells cultured with (A) 0.5% DMSO (control), and (B) 31.25 µg/ml and (C) 62.5 µg/ml of petroleum ether extract of Chenopodium album L. for 24 h using flow cytometry after the cells were stained by <t>propidium</t> iodide. (D) Histograms present the percentage of each cell populations at the different cell cycle stage following treatment. Data presented as the mean ± standard deviation of at least three experiments. **P
    Propidium Iodide Pi, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 1003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 1151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend propidium iodide
    Neisseria gonorrhoeae induces cell death in monocyte-derived macrophages. MDMs were stimulated with medium alone (unstimulated), LPS (100 ng/mL, Escherichia coli O111:B4) and 5 mM ATP (LPS/ATP), 3 μM staurosporine (STS), formalin-fixed bacteria MOI 100 (FF MOI 100), or live bacteria MOI 10 or 100 of strain FA1090B. (A) Cell death was determined by trypan exclusion at 1, 6, 12, and 24 hours. (B) Cell death at 6 hours was confirmed by flow cytometry as the percent of total MDMs staining positive for <t>propidium</t> iodide. The graphs depict the mean ± SEM from 8 donors; 1-way ANOVA with a Dunnett multiple comparisons posttest was performed at each time point (* P
    Propidium Iodide, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Olympus propidium iodide
    Ca 2+ alleviation of H + and STOP1 . Seedlings grown for 5 d in a control solution (pH 5.5) were soaked in a solution containing 200 or 400 μ m CaCl 2 (pH 4.7) for 90 min and then stained with <t>propidium</t> iodide.
    Propidium Iodide, supplied by Olympus, used in various techniques. Bioz Stars score: 92/100, based on 1099 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Keygen Biotech propidium iodide pi
    Effects of indirubin and arsenic disulfide (As 2 S 2 ), alone or in combination, on the apoptosis of LY8 cells. LY8 cells were incubated with different doses of indirubin (1, 5, 10, 15 and 20 μM) and also 10 μM As 2 S 2 and 20 μM indirubin, alone or in combination, for 48 h. (A) The apoptotic rate was determined using Annexin V-fluorescein isothiocyanate <t>(FITC)/propidium</t> iodide (PI) dual staining followed by flow cytometric analysis. The lower left quadrant (Q4) indicates the percentage of viable cells (Annexin V-FITC- and PI-negative), the upper left quadrant (Q1) indicates the percentage of early apoptotic cells (Annexin V-FITC-positive and PI-negative) and the upper right quadrant (Q2) indicates the percentage of late apoptotic cells (Annexin V-FITC- and PI-positive). (B) There were no significant effects on the rate of cell apoptosis of the LY8 cells following incubation with different doses of indirubin. However, significant differences were evident between the As 2 S 2 -treated group and the combination-treated group. Values are expressed as the mean ± standard deviation. ** P
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    Becton Dickinson propidium iodide solution
    Cell cycle analysis. GL15 (A) , U87MG (B) and U251 (C) cells were treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin for 24 h and 48 h respectively. Cell distribution in G0/G1, S and G2/M phases was analyzed by flow cytometry using <t>propidium</t> iodide DNA staining. Legend: *Any inhibitor/control, # PP242/wortmannin, ≈ PP242/rapamycin, ∧ rapamycin/wortmannin ( *,#,∧,≈ p
    Propidium Iodide Solution, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam propidium iodide
    NCS‐01 cells, in part, employ filopodia‐mediated long distance rescue of cultured primary rat cortical cells against oxygen glucose deprivation (OGD). Primary rat cortical cells were subjected to OGD and reperfusion then cocultured with NCS‐01 cells at different distances, as depicted by procedural timeline and diagram (A and A′). <t>Propidium</t> iodide and N‐cadherin staining was conducted for the different distances: (B and B′) 1.68 mm, (C and C′) 1.80 mm, (D and D′) 1.92 mm, and (E and E′) 2.04 mm. Propidium iodide staining (red) reveals fewer dead primary rat cortical cells (indicated by red color) at closer distances than at farther distances between upper and lower chambers (B‐E). N‐cadherin staining (green) indicates the presence of filopodia extending from NCS‐01 cells (B′‐E′) Scale bar = 50 μm. Quantitative analysis of primary rat cortical neurons reveals that cell viability correlates inversely with increased distance between NCS‐01 cells and primary rat cortical neurons, when measured by trypan blue (TB) assay (F). Similarly, MTT assay reveals that greater mitochondrial activity correlates inversely with greater distance between NCS‐01 cells and primary rat cortical neurons (G). Significance depicted as a‐d at P
    Propidium Iodide, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ATR signaling is activated by A3A expression. (A) A3A activates DNA damage checkpoints. Inducible cell lines were treated with doxycycline for 24 hours and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk2 (T68), phosphorylated Chk1 (S345 and S317), phosphorylated ATM (S1981), Chk2, Chk1, ATM, and ATR. GAPDH was used as a loading control. (B) A3A expression results in cell cycle arrest. Inducible cells lines were treated with doxycycline for 24 or 36 hours, then pulsed with BrdU for 40 minutes prior to harvest. Cells were fixed, stained with anti-BrdU antibody and 7-AAD, and analyzed by flow cytometry. The percentage of cells in each gate are indicated. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 36 hours post-induction. (C) A3A activates ATR signaling in ATM-deficient cells. Inducible AT22IJE-A3A cells with mutant ATM or wild-type ATM were treated with doxycycline to induce A3A expression and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk1 (S345 and S317), Chk1, phosphorylated ATM (S1981), ATM, and ATR. (D) Cell-cycle progression following A3A induction in AT22IJE cells with mutant ATM or wild-type ATM was determined by propidium iodide staining. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 24 hours after treatment, which was similar to results obtained 48 hours after treatment. Results are representative of 3 independent experiments.

    Journal: Cell Cycle

    Article Title: APOBEC3A damages the cellular genome during DNA replication

    doi: 10.1080/15384101.2016.1152426

    Figure Lengend Snippet: ATR signaling is activated by A3A expression. (A) A3A activates DNA damage checkpoints. Inducible cell lines were treated with doxycycline for 24 hours and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk2 (T68), phosphorylated Chk1 (S345 and S317), phosphorylated ATM (S1981), Chk2, Chk1, ATM, and ATR. GAPDH was used as a loading control. (B) A3A expression results in cell cycle arrest. Inducible cells lines were treated with doxycycline for 24 or 36 hours, then pulsed with BrdU for 40 minutes prior to harvest. Cells were fixed, stained with anti-BrdU antibody and 7-AAD, and analyzed by flow cytometry. The percentage of cells in each gate are indicated. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 36 hours post-induction. (C) A3A activates ATR signaling in ATM-deficient cells. Inducible AT22IJE-A3A cells with mutant ATM or wild-type ATM were treated with doxycycline to induce A3A expression and analyzed by Western blot using antibodies to HA, γH2AX, phosphorylated Chk1 (S345 and S317), Chk1, phosphorylated ATM (S1981), ATM, and ATR. (D) Cell-cycle progression following A3A induction in AT22IJE cells with mutant ATM or wild-type ATM was determined by propidium iodide staining. Accompanying chart shows the fraction of cells in G1, S, and G2/M phase at 24 hours after treatment, which was similar to results obtained 48 hours after treatment. Results are representative of 3 independent experiments.

    Article Snippet: HepaRG, U2OS, and AT22IJE cells were grown in the presence or absence of doxycycline (0.1-2 μg/ml, Clontech), fixed in 70% ice-cold ethanol, washed in PBS, and resuspended in staining solution containing 20 μg/ml propidium iodide (Sigma) and 200 μg/ml RNAse A (Roche).

    Techniques: Expressing, Western Blot, Staining, Flow Cytometry, Cytometry, Mutagenesis

    Yes inhibitors block NF2 -deficient PRCC cell cycle Quantification of UOK275 ( A ) and HEK293 ( B ) cell cycle phases by BrdU and propidium iodide (PI) staining. The panels display the position of gates used to quantify cells in G0-G1, S or G2-M phases after 18 hours of treatment with DMSO (control), or dasatinib (50 nM or 500 nM). ( C ) Quantification of cells in the different cell cycle phases in HEK293, UOK275, UOK342 and ACHN after DMSO or dasatinib treatment (average of 3 distinct experiments).

    Journal: Oncotarget

    Article Title: Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers

    doi: 10.18632/oncotarget.24112

    Figure Lengend Snippet: Yes inhibitors block NF2 -deficient PRCC cell cycle Quantification of UOK275 ( A ) and HEK293 ( B ) cell cycle phases by BrdU and propidium iodide (PI) staining. The panels display the position of gates used to quantify cells in G0-G1, S or G2-M phases after 18 hours of treatment with DMSO (control), or dasatinib (50 nM or 500 nM). ( C ) Quantification of cells in the different cell cycle phases in HEK293, UOK275, UOK342 and ACHN after DMSO or dasatinib treatment (average of 3 distinct experiments).

    Article Snippet: DNA was stained for 30 minutes with IFA buffer with of 50 μg/mL propidium iodide (PI) (Sigma-Aldrich #P4864) and 5 μg/mL RNase A (Sigma #R4642).

    Techniques: Blocking Assay, Staining

    Loss of TKTL1 expression changes cell cycle distribution of melanoma cells. Cell cycle phases were determined by propidium iodide staining of melanoma cells and subsequent flow cytometric analysis. A representative histogram of cell cycle analysis of LM-MEL-59 is shown after a control siRNA treatment and b TKTL1 siRNA treatment. Analysis of percentage of cells in e G0-G1 phase and S phase cell after treatment of LM-MEL-59 with TKTL1 or control siRNA. Values are ± SD of three experiments in triplicate (*, p

    Journal: BMC Cancer

    Article Title: Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells

    doi: 10.1186/s12885-016-2185-5

    Figure Lengend Snippet: Loss of TKTL1 expression changes cell cycle distribution of melanoma cells. Cell cycle phases were determined by propidium iodide staining of melanoma cells and subsequent flow cytometric analysis. A representative histogram of cell cycle analysis of LM-MEL-59 is shown after a control siRNA treatment and b TKTL1 siRNA treatment. Analysis of percentage of cells in e G0-G1 phase and S phase cell after treatment of LM-MEL-59 with TKTL1 or control siRNA. Values are ± SD of three experiments in triplicate (*, p

    Article Snippet: TKTL1 was over-expressed or depleted in melanoma cells for 48 h. Cells were stained with 200 μl propidium iodide solution consisting of 50 μg/ml propidium iodide, 0.1 mg/ml RNAse A and 0.05 % Triton-X (all Sigma) in PBS.

    Techniques: Expressing, Staining, Flow Cytometry, Cell Cycle Assay

    ELK1 protein induces G 1 arrest and apoptosis. ( A ) Cell-cycle progression of CL1-5 cells overexpressing ELK1. Cells were transfected with ELK1 or mock vector, harvested, fixed with ethanol, and stained with propidium iodide. DNA content was measured by

    Journal: RNA

    Article Title: Negative feedback regulation of AXL by miR-34a modulates apoptosis in lung cancer cells

    doi: 10.1261/rna.052571.115

    Figure Lengend Snippet: ELK1 protein induces G 1 arrest and apoptosis. ( A ) Cell-cycle progression of CL1-5 cells overexpressing ELK1. Cells were transfected with ELK1 or mock vector, harvested, fixed with ethanol, and stained with propidium iodide. DNA content was measured by

    Article Snippet: At harvest, cells were collected and washed twice with PBS, resuspended in 1 mL of PBS, fixed by adding 4 mL of 100% ethanol at −20°C for 2–24 h, and then stained with a propidium iodide (PI) solution containing 50 μg/mL PI and 50 μg/mL RNase (Sigma-Aldrich) in PBS at room temperature for 30 min in the dark.

    Techniques: Transfection, Plasmid Preparation, Staining

    DAPK1 Regulates Apoptosis in Lymphoid Cells (A) DAPK1 expression in Jurkat cells stably transfected with either vector alone, DAPK1 siRNA-A, or DAPK1 siRNA-C as measured by western blot. Tubulin expression served as a control. (B) Percent live cells were measured in Jurkat cells stably transfected with vector alone or DAPK1 siRNA-C, treated with activating-Fas antibody (100 ng/ml). After 16 hr, cells were harvested and suspended in binding buffer with annexin V-FITC and propidium iodide, followed by flow cytometry to assess cell death. Error bars indicate SD. (C) p53 expression in cells treated with either no, 50 ng/ml, or 100 ng/ml Fas-activating antibody.

    Journal: Cell

    Article Title: Downregulation of Death-Associated Protein Kinase 1 (DAPK1) in Chronic Lymphocytic Leukemia

    doi: 10.1016/j.cell.2007.03.043

    Figure Lengend Snippet: DAPK1 Regulates Apoptosis in Lymphoid Cells (A) DAPK1 expression in Jurkat cells stably transfected with either vector alone, DAPK1 siRNA-A, or DAPK1 siRNA-C as measured by western blot. Tubulin expression served as a control. (B) Percent live cells were measured in Jurkat cells stably transfected with vector alone or DAPK1 siRNA-C, treated with activating-Fas antibody (100 ng/ml). After 16 hr, cells were harvested and suspended in binding buffer with annexin V-FITC and propidium iodide, followed by flow cytometry to assess cell death. Error bars indicate SD. (C) p53 expression in cells treated with either no, 50 ng/ml, or 100 ng/ml Fas-activating antibody.

    Article Snippet: Apoptosis and Flow Cytometric Studies Jurkat cells stably transfected with pRS vector control or pRS-DAPK siRNA C were treated with 100 ng/ml anti-Fas (human activating, clone CH11) antibody (Upstate Biotechnology, Lake Placid, NY) for 16 hr and resuspended in binding buffer containing annexin V-fluorescein isothiocyanate (FITC) and propidium iodide according to the supplier's instuctions (BD Biosciences, San Diego, CA), and assessed by flow cytometry using a Beckman-Coulter model EPICS XL cytometer (Beckman-Coulter, Miami, FL).

    Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Flow Cytometry, Cytometry

    Cx43 expression and localization during fibroblast to myofibroblast transition and related PRP effects. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. The cells cultured in proliferation medium (PM) were used as control undifferentiated cells. ( A ) RT-PCR analysis of Cx43 expression in the indicated experimental conditions. Representative agarose gel is shown. The densitometric analysis of the bands normalized to β-actin is reported in the histogram. ( B ) Western Blotting analysis of Cx43 expression. Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( C – E ) Representative superimposed differential interference contrast (DIC, grey) and confocal fluorescence images of the cells immunostained with antibodies against Cx43 (green) and counterstained with propidium iodide (PI, red) to label nuclei. Scale bar: 30 µm. Scale bar in the inset in D: 15 µm. Arrows indicate the localization of Cx43 at the membrane level of two adjacent cells. ( F ) Histogram showing the densitometric analysis of the intensity of the Cx43 fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (12). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Journal: Cells

    Article Title: Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1–Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis

    doi: 10.3390/cells9051199

    Figure Lengend Snippet: Cx43 expression and localization during fibroblast to myofibroblast transition and related PRP effects. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. The cells cultured in proliferation medium (PM) were used as control undifferentiated cells. ( A ) RT-PCR analysis of Cx43 expression in the indicated experimental conditions. Representative agarose gel is shown. The densitometric analysis of the bands normalized to β-actin is reported in the histogram. ( B ) Western Blotting analysis of Cx43 expression. Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( C – E ) Representative superimposed differential interference contrast (DIC, grey) and confocal fluorescence images of the cells immunostained with antibodies against Cx43 (green) and counterstained with propidium iodide (PI, red) to label nuclei. Scale bar: 30 µm. Scale bar in the inset in D: 15 µm. Arrows indicate the localization of Cx43 at the membrane level of two adjacent cells. ( F ) Histogram showing the densitometric analysis of the intensity of the Cx43 fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (12). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Article Snippet: In some experiments the fixed cells were incubated with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, 1:100; Molecular Probes) for 10 min at room temperature, which binds glycoconjugates present on cell membranes, or counterstained with propidium iodide (PI, 1:30 for 30 s; Molecular Probes), to detect nuclei.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Fluorescence

    Evaluation of the effects PRP on fibroblast to myofibroblast transition and of the involvement of GJs: α-sma expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. Cells cultured in proliferation medium (PM) served as control undifferentiated cells. In parallel experiments, fibroblasts were cultured in PM or in DM in the presence of heptanol (HEPT), a common GJ channel blocker, in the presence or absence of PRP for 72 h. ( A , B ) Western Blotting analysis of α-sma expression. ( A ) Representative Blot. ( B ) Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( C – H ) Representative confocal fluorescence images of the cells immunostained with antibodies against α-sma (green) and counterstained with propidium iodide (PI) to detect nuclei. Scale bar: 50 µm. ( I ) Histogram showing the densitometric analysis of the intensity of the α-sma fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (10). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Journal: Cells

    Article Title: Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1–Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis

    doi: 10.3390/cells9051199

    Figure Lengend Snippet: Evaluation of the effects PRP on fibroblast to myofibroblast transition and of the involvement of GJs: α-sma expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. Cells cultured in proliferation medium (PM) served as control undifferentiated cells. In parallel experiments, fibroblasts were cultured in PM or in DM in the presence of heptanol (HEPT), a common GJ channel blocker, in the presence or absence of PRP for 72 h. ( A , B ) Western Blotting analysis of α-sma expression. ( A ) Representative Blot. ( B ) Histogram showing the densitometric analysis of the bands normalized to α-tubulin. ( C – H ) Representative confocal fluorescence images of the cells immunostained with antibodies against α-sma (green) and counterstained with propidium iodide (PI) to detect nuclei. Scale bar: 50 µm. ( I ) Histogram showing the densitometric analysis of the intensity of the α-sma fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (10). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Article Snippet: In some experiments the fixed cells were incubated with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, 1:100; Molecular Probes) for 10 min at room temperature, which binds glycoconjugates present on cell membranes, or counterstained with propidium iodide (PI, 1:30 for 30 s; Molecular Probes), to detect nuclei.

    Techniques: Expressing, Cell Culture, Western Blot, Fluorescence

    Effects of PRP on fibroblast to myofibroblast transition: Cell morphology and type-1 collagen expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 72 h. The cells cultured in proliferation medium (PM) served as control undifferentiated cells. ( A – F ) Representative confocal fluorescence images of the cells ( A – C ) stained with Alexa Fluor 488-conjugated WGA (green) to reveal the plasma membrane and ( D – F ) immunostained with antibodies against type-1 collagen (green) and counterstained with propidium iodide (PI), to label nuclei. Scale bar: 50 µm. ( G ) Histogram showing the densitometric analysis of the intensity of type-1 collagen fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (10). Data are reported as mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Journal: Cells

    Article Title: Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1–Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis

    doi: 10.3390/cells9051199

    Figure Lengend Snippet: Effects of PRP on fibroblast to myofibroblast transition: Cell morphology and type-1 collagen expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 72 h. The cells cultured in proliferation medium (PM) served as control undifferentiated cells. ( A – F ) Representative confocal fluorescence images of the cells ( A – C ) stained with Alexa Fluor 488-conjugated WGA (green) to reveal the plasma membrane and ( D – F ) immunostained with antibodies against type-1 collagen (green) and counterstained with propidium iodide (PI), to label nuclei. Scale bar: 50 µm. ( G ) Histogram showing the densitometric analysis of the intensity of type-1 collagen fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm 2 for each confocal stack (10). Data are reported as mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: * p

    Article Snippet: In some experiments the fixed cells were incubated with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, 1:100; Molecular Probes) for 10 min at room temperature, which binds glycoconjugates present on cell membranes, or counterstained with propidium iodide (PI, 1:30 for 30 s; Molecular Probes), to detect nuclei.

    Techniques: Expressing, Cell Culture, Fluorescence, Staining, Whole Genome Amplification

    Development of vasculature scaffold in the RMS. A , Micrographs displaying BVs in the RMS at different developmental periods. Blood vessels were labeled by PECAM immunostaining, and RMS was visualized by dense DAPI labeling. Note that the first parallel

    Journal: The Journal of Neuroscience

    Article Title: Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling

    doi: 10.1523/JNEUROSCI.5531-11.2012

    Figure Lengend Snippet: Development of vasculature scaffold in the RMS. A , Micrographs displaying BVs in the RMS at different developmental periods. Blood vessels were labeled by PECAM immunostaining, and RMS was visualized by dense DAPI labeling. Note that the first parallel

    Article Snippet: We distinguished the RMS by DAPI (Invitrogen) nuclear counterstaining, according to the manufacturer's instructions.

    Techniques: Labeling, Immunostaining

    RMS-SVZ astrocytes can be specifically targeted in vivo . A , Confocal images demonstrating the high infection level after GFAP-GFP VEGF miRNA injection into two sites of P3 RMS. Infected cells are in green, and RMS was delimited by DAPI labeling (blue).

    Journal: The Journal of Neuroscience

    Article Title: Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling

    doi: 10.1523/JNEUROSCI.5531-11.2012

    Figure Lengend Snippet: RMS-SVZ astrocytes can be specifically targeted in vivo . A , Confocal images demonstrating the high infection level after GFAP-GFP VEGF miRNA injection into two sites of P3 RMS. Infected cells are in green, and RMS was delimited by DAPI labeling (blue).

    Article Snippet: We distinguished the RMS by DAPI (Invitrogen) nuclear counterstaining, according to the manufacturer's instructions.

    Techniques: In Vivo, Infection, Injection, Labeling

    HsLin06_18-FITC uptake and membrane permeabilization in Candida albicans biofilms treated with the [caspofungin + HsLin06_18-FITC] combination. Confocal microscope images of 24 h treated C. albicans biofilms with 0.625 μM caspofungin (CASPO) and/or 0.5 μM HsLin06_18-FITC and 30 μg/mL propidium iode (PI). Bar: 5 μm.

    Journal: Frontiers in Microbiology

    Article Title: A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms

    doi: 10.3389/fmicb.2017.02051

    Figure Lengend Snippet: HsLin06_18-FITC uptake and membrane permeabilization in Candida albicans biofilms treated with the [caspofungin + HsLin06_18-FITC] combination. Confocal microscope images of 24 h treated C. albicans biofilms with 0.625 μM caspofungin (CASPO) and/or 0.5 μM HsLin06_18-FITC and 30 μg/mL propidium iode (PI). Bar: 5 μm.

    Article Snippet: Exponentially growing cells were exposed to caspofungin (0.01 μM), HsLin06_18-FITC (4.6 μM) or its combination (as described in the material and methods section “fungicidal activity assay”), and to propidium iodide (PI) (2 μg/mL), after which they were subjected to flow cytometry on a BD Influx™ cell sorter.

    Techniques: Microscopy

    Kinetics of HsLin06_18-FITC uptake and membrane permeabilization in planktonic Candida albicans cells treated with (A) 4.6 μM HsLin06_18-FITC (HsL-F), (B) 0.01 μM caspofungin (CASPO) or (C) its combination as well as 2 μg/mL propidium iodide (PI), determined via flow cytometry. For all treatments, the % of cells is presented that: only have permeabilized membranes (gray), both have permeabilized membranes and HsLin06_18-FITC associated to their surface or internalized (orange) or only HsLin06_18-FITC associated to their surface or internalized (black). Data are means ± SEM in presented for n = 3 independent experiments. To analyse significant differences in the size of the subpopulations between the t0 and other time points, one-way ANOVA followed by Dunnett multiple comparison was performed, with brackets (in the color of the corresponding subpopulation) representing significance for that subpopulation. Only the primary time point that is significantly different from t0 is presented, with * representing P

    Journal: Frontiers in Microbiology

    Article Title: A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms

    doi: 10.3389/fmicb.2017.02051

    Figure Lengend Snippet: Kinetics of HsLin06_18-FITC uptake and membrane permeabilization in planktonic Candida albicans cells treated with (A) 4.6 μM HsLin06_18-FITC (HsL-F), (B) 0.01 μM caspofungin (CASPO) or (C) its combination as well as 2 μg/mL propidium iodide (PI), determined via flow cytometry. For all treatments, the % of cells is presented that: only have permeabilized membranes (gray), both have permeabilized membranes and HsLin06_18-FITC associated to their surface or internalized (orange) or only HsLin06_18-FITC associated to their surface or internalized (black). Data are means ± SEM in presented for n = 3 independent experiments. To analyse significant differences in the size of the subpopulations between the t0 and other time points, one-way ANOVA followed by Dunnett multiple comparison was performed, with brackets (in the color of the corresponding subpopulation) representing significance for that subpopulation. Only the primary time point that is significantly different from t0 is presented, with * representing P

    Article Snippet: Exponentially growing cells were exposed to caspofungin (0.01 μM), HsLin06_18-FITC (4.6 μM) or its combination (as described in the material and methods section “fungicidal activity assay”), and to propidium iodide (PI) (2 μg/mL), after which they were subjected to flow cytometry on a BD Influx™ cell sorter.

    Techniques: Flow Cytometry, Cytometry

    Naegleria fowleri trophozoites incubated with IC 90 of STS for 24 h ( D – F ). Negative control ( A – C ). Hoechst stain is different in control cells, where no fluorescence is observed ( B ), and in treated cells, where the nuclei are intense blue ( E ). Red fluorescence corresponds to the propidium iodide stain ( C , F ). Visible channel ( A , D ); Hoechst channel ( B , E ), and propidium iodide channel ( C , F ). Images (40×) are representative of the cell population observed in the performed experiments. Images were obtained using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. Obtained results were similar for both N. fowleri type strains.

    Journal: Microorganisms

    Article Title: Evaluation of Indolocarbazoles from Streptomyces sanyensis as a Novel Source of Therapeutic Agents against the Brain-Eating Amoeba Naegleria fowleri

    doi: 10.3390/microorganisms8050789

    Figure Lengend Snippet: Naegleria fowleri trophozoites incubated with IC 90 of STS for 24 h ( D – F ). Negative control ( A – C ). Hoechst stain is different in control cells, where no fluorescence is observed ( B ), and in treated cells, where the nuclei are intense blue ( E ). Red fluorescence corresponds to the propidium iodide stain ( C , F ). Visible channel ( A , D ); Hoechst channel ( B , E ), and propidium iodide channel ( C , F ). Images (40×) are representative of the cell population observed in the performed experiments. Images were obtained using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. Obtained results were similar for both N. fowleri type strains.

    Article Snippet: Detection of DNA Condensation The double-stain apoptosis detection kit constituted by Hoechst 33342 and propidium iodide (PI) (Life Technologies, Madrid.

    Techniques: Incubation, Negative Control, Staining, Fluorescence, Imaging

    RUNX1 silencing deregulates breast cancer cell mitosis. RUNX1 was knocked down in MCF7 cells either constitutively ( a (left) and b ) or conditionally upon treatment with dox ( a (right), c , d g and h ). Cells were maintained in medium supplemented with either complete serum ( a , c , d , f – h ) or CSS ( b ). ( a , b ) Cell cycle profiles were obtained by FACS analysis of propidium iodide-stained cells. In b , cells were treated with either vehicle control (EtOH) or estradiol (E2) for 48 h as indicated. * P

    Journal: Nature Communications

    Article Title: RUNX1 prevents oestrogen-mediated AXIN1 suppression and β-catenin activation in ER-positive breast cancer

    doi: 10.1038/ncomms10751

    Figure Lengend Snippet: RUNX1 silencing deregulates breast cancer cell mitosis. RUNX1 was knocked down in MCF7 cells either constitutively ( a (left) and b ) or conditionally upon treatment with dox ( a (right), c , d g and h ). Cells were maintained in medium supplemented with either complete serum ( a , c , d , f – h ) or CSS ( b ). ( a , b ) Cell cycle profiles were obtained by FACS analysis of propidium iodide-stained cells. In b , cells were treated with either vehicle control (EtOH) or estradiol (E2) for 48 h as indicated. * P

    Article Snippet: The cells were incubated for 16 h in medium containing 2 mM thymidine (Sigma-Aldrich), released for 8–12 h in DMEM containing 10% FBS, and then incubated again in 2 mM thymidine for 16–18 h. For G2/M synchronization, the cells were released from the double-thymidine block for 4 h before treatment with 100 nM nocodazole (Sigma-Aldrich) for 14 h. Percentage of cells in the various phases of the cell cycle was quantified by propidium iodide staining and flow cytometry using LSRII flow cytometer (BD Biosciences) and the Modfit LT SynchWizard Tool (Verity Software House).

    Techniques: FACS, Staining

    The adipocytokines leptin, adiponectin, Nampt and NMN have no direct effects on beta-cell survival in INS-1E cells. INS-1E cells were kept under serum-free conditions 24 h before and during the 48 h experiment. ( A,B ) INS-1E cells were exposed to cytokines ( A : IL-1β, IFN-γ or TNFα) or adipocytokines ( B : adiponectin, leptin, Nampt, NMN) at the indicated concentrations for 48 h and cell viability was measured by WST-1 assay. Data are shown as means ±SEM of 3 independent experiments performed in triplicates. Statistical analyses were performed by one-way ANOVA with Bonferroni’s Multiple Comparison Test as posthoc test. C,D : INS-1E cells were exposed to adipocytokines (adiponectin 167 ng/ml, leptin 200 ng/ml, Nampt 2.5 ng/ml, NMN 100 µM) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ) for 48 h. Cytotoxicity ( C ) was analyzed by measuring the release of adenylate kinase into the supernatant and ( D ) apoptosis was measured by FITC-annexinV (An) and propidium iodide (PI) staining and subsequent flow cytometric analysis of An-positive and double An/PI positive cells. Results were expressed relative to cells exposed to serum free medium (con) and as means ±SEM of three independent experiments performed in triplicates. E,F : INS-1E cells were exposed to a cytokine combination (IL/IF; 10 ng/ml IL-1β+10 ng/ml IFN-γ) ( E ) or 0.25 mM palmitate (pal) ( F ) for 48 h in the absence or presence of the adipocytokines (167 ng/ml adiponectin, 200 ng/ml leptin, 2.5 ng/ml Nampt) and induction of apoptosis was measured by An/PI staining and flow cytometric analysis. Data are shown as means ±SEM of triplicates of three independent experiments. Statistical analyses were performed by student´s t-test. G: INS-1E cells were exposed to the adipocytokines adiponectin (167 ng/ml), leptin (200 ng/ml) or Nampt (2.5 ng/ml) or a combination of camptothecin (2 µM) and etoposide (85 µM; CE, upper and lower panel ) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ, middle and lower panel ). Western blot analyses were performed for full length and cleaved caspase-3 ( upper panel ), phospho-NF-κB p65 (Ser536) and NF-κB p65 ( middle panel ) and phospho-p53 (Ser15) ( lower panel ). GAPDH or beta-actin were used as loading control. All panels show one typical blot out of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: The Adipocytokine Nampt and Its Product NMN Have No Effect on Beta-Cell Survival but Potentiate Glucose Stimulated Insulin Secretion

    doi: 10.1371/journal.pone.0054106

    Figure Lengend Snippet: The adipocytokines leptin, adiponectin, Nampt and NMN have no direct effects on beta-cell survival in INS-1E cells. INS-1E cells were kept under serum-free conditions 24 h before and during the 48 h experiment. ( A,B ) INS-1E cells were exposed to cytokines ( A : IL-1β, IFN-γ or TNFα) or adipocytokines ( B : adiponectin, leptin, Nampt, NMN) at the indicated concentrations for 48 h and cell viability was measured by WST-1 assay. Data are shown as means ±SEM of 3 independent experiments performed in triplicates. Statistical analyses were performed by one-way ANOVA with Bonferroni’s Multiple Comparison Test as posthoc test. C,D : INS-1E cells were exposed to adipocytokines (adiponectin 167 ng/ml, leptin 200 ng/ml, Nampt 2.5 ng/ml, NMN 100 µM) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ) for 48 h. Cytotoxicity ( C ) was analyzed by measuring the release of adenylate kinase into the supernatant and ( D ) apoptosis was measured by FITC-annexinV (An) and propidium iodide (PI) staining and subsequent flow cytometric analysis of An-positive and double An/PI positive cells. Results were expressed relative to cells exposed to serum free medium (con) and as means ±SEM of three independent experiments performed in triplicates. E,F : INS-1E cells were exposed to a cytokine combination (IL/IF; 10 ng/ml IL-1β+10 ng/ml IFN-γ) ( E ) or 0.25 mM palmitate (pal) ( F ) for 48 h in the absence or presence of the adipocytokines (167 ng/ml adiponectin, 200 ng/ml leptin, 2.5 ng/ml Nampt) and induction of apoptosis was measured by An/PI staining and flow cytometric analysis. Data are shown as means ±SEM of triplicates of three independent experiments. Statistical analyses were performed by student´s t-test. G: INS-1E cells were exposed to the adipocytokines adiponectin (167 ng/ml), leptin (200 ng/ml) or Nampt (2.5 ng/ml) or a combination of camptothecin (2 µM) and etoposide (85 µM; CE, upper and lower panel ) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ, middle and lower panel ). Western blot analyses were performed for full length and cleaved caspase-3 ( upper panel ), phospho-NF-κB p65 (Ser536) and NF-κB p65 ( middle panel ) and phospho-p53 (Ser15) ( lower panel ). GAPDH or beta-actin were used as loading control. All panels show one typical blot out of three independent experiments. *p

    Article Snippet: Apoptosis in INS-1E cells was assessed by FITC-AnnexinV (An) and propidium iodide (PI) staining (BD, Heidelberg, Germany) and flow cytometric analysis (BD FACSCalibur).

    Techniques: WST-1 Assay, Staining, Flow Cytometry, Western Blot

    miR-18a induces DNA damage. a Expression of γ-H2AX as measured by immunofluorescence in Raji cells; EBV-positive Raji cells were stained with anti-γ-H2AX and DAPI. Expression of γ-H2AX is indicated as green loci. DAPI was used to stain the cell nuclei. The merge images present the DAPI and FITC as blue and green, respectively. b Expression of γ-H2AX as measured by western blotting in EBV-positive or -negative cells. c Detection of DNA damage after transfection of miR-18a. The comet assay was applied. Cells were electrophoresed in agarose gels on a coverslip and were stained with propidium iodide. Labeled DNA was visualized under a fluorescence microscope. d Detection of DNA damage after transfection of miR-18a in EBV-negative and -positive BJAB cells. Magnification, × 100. e Graphic presentation of all chromosomal changes. Cells transfected with miR-18a and mimics negative control were analyzed by comparative genomic hybridization array (Array-CGH). The regions of DNA gain (blue) and loss (red) are shown

    Journal: BMC Cancer

    Article Title: miR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

    doi: 10.1186/s12885-018-5205-9

    Figure Lengend Snippet: miR-18a induces DNA damage. a Expression of γ-H2AX as measured by immunofluorescence in Raji cells; EBV-positive Raji cells were stained with anti-γ-H2AX and DAPI. Expression of γ-H2AX is indicated as green loci. DAPI was used to stain the cell nuclei. The merge images present the DAPI and FITC as blue and green, respectively. b Expression of γ-H2AX as measured by western blotting in EBV-positive or -negative cells. c Detection of DNA damage after transfection of miR-18a. The comet assay was applied. Cells were electrophoresed in agarose gels on a coverslip and were stained with propidium iodide. Labeled DNA was visualized under a fluorescence microscope. d Detection of DNA damage after transfection of miR-18a in EBV-negative and -positive BJAB cells. Magnification, × 100. e Graphic presentation of all chromosomal changes. Cells transfected with miR-18a and mimics negative control were analyzed by comparative genomic hybridization array (Array-CGH). The regions of DNA gain (blue) and loss (red) are shown

    Article Snippet: Finally, the cells were stained with 10 μg/ml propidium iodide (Beyotime Biotechnology, ST511), and individual cells were viewed using an Olympus FSX100 fluorescence microscope.

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Transfection, Single Cell Gel Electrophoresis, Labeling, Fluorescence, Microscopy, Negative Control, Hybridization

    BaF3/ITD-R and MV4-11-R cells are highly resistant to sorafenib. a BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with the indicated concentrations of sorafenib for 48 h and subjected to an Annexin-V/PI assay. The percentage values indicate the total cell death populations. b BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with various concentrations of sorafenib for 72 h and subjected to an MTS assay. The numbers on the curves indicate the IC 50 of sorafenib in each cell line. PI propidium iodide

    Journal: Cancer Communications

    Article Title: Metabolic reprogramming and redox adaptation in sorafenib-resistant leukemia cells: detected by untargeted metabolomics and stable isotope tracing analysis

    doi: 10.1186/s40880-019-0362-z

    Figure Lengend Snippet: BaF3/ITD-R and MV4-11-R cells are highly resistant to sorafenib. a BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with the indicated concentrations of sorafenib for 48 h and subjected to an Annexin-V/PI assay. The percentage values indicate the total cell death populations. b BaF3/ITD, BaF3/ITD-R, MV4-11 and MV4-11-R cells were treated with various concentrations of sorafenib for 72 h and subjected to an MTS assay. The numbers on the curves indicate the IC 50 of sorafenib in each cell line. PI propidium iodide

    Article Snippet: For cell cycle analysis, cells were serum starved overnight and stimulated with complete medium for 6 h. Cells were fixed in 70% ethanol, stained with propidium iodide and measured by flow cytometer.

    Techniques: MTS Assay

    Effect of Ia on P. aeruginosa biofilms. ( a ) Untreated GFP-labelled PA14 biofilm; ( b ) GFP- labelled PA14 biofilm grown with Ia at 8 µM; ( c ) GFP-labelled PA14 biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( d ) GFP-labelled PA14 biofilm grown with 8 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( e ) Biomass quantitation of PA14 biofilms; ( f ) Untreated GFP-labelled PAO1-L biofilm; ( g ) GFP-labelled PAO1-L biofilm grown with Ia at 34 µM; ( h ) GFP-labelled PAO1-L biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( i ) GFP-labelled PAO1-L biofilm grown with 34 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of incubation; ( j ) Biomass quantitation of PAO1-L biofilms. Dead cells and extracellular DNA were stained with propidium iodide (PI). Three-dimensional (3D) sections and cross sections are shown. Scale bars represent 100 µm.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: In Silico and in Vitro-Guided Identification of Inhibitors of Alkylquinolone-Dependent Quorum Sensing in Pseudomonas aeruginosa

    doi: 10.3390/molecules23020257

    Figure Lengend Snippet: Effect of Ia on P. aeruginosa biofilms. ( a ) Untreated GFP-labelled PA14 biofilm; ( b ) GFP- labelled PA14 biofilm grown with Ia at 8 µM; ( c ) GFP-labelled PA14 biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( d ) GFP-labelled PA14 biofilm grown with 8 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( e ) Biomass quantitation of PA14 biofilms; ( f ) Untreated GFP-labelled PAO1-L biofilm; ( g ) GFP-labelled PAO1-L biofilm grown with Ia at 34 µM; ( h ) GFP-labelled PAO1-L biofilm, treated with 100 µg/mL tobramycin for 4 h after 16 h of growth; ( i ) GFP-labelled PAO1-L biofilm grown with 34 µM Ia and treated with 100 µg/mL tobramycin for 4 h after 16 h of incubation; ( j ) Biomass quantitation of PAO1-L biofilms. Dead cells and extracellular DNA were stained with propidium iodide (PI). Three-dimensional (3D) sections and cross sections are shown. Scale bars represent 100 µm.

    Article Snippet: Tobramycin and propidium iodide were added to the 15 h-old cultures at concentrations of 100 µg/mL and 2 µM, respectively, followed by further incubation for 4 h. Coverslips were examined under a Laser Scanning Fluorescent Microscope (LSM2, Zeiss, Oberkochen, Germany).

    Techniques: IA, Quantitation Assay, Incubation, Staining

    Cell cycle analysis of A549 cells cultured with (A) 0.5% DMSO (control), and (B) 31.25 µg/ml and (C) 62.5 µg/ml of petroleum ether extract of Chenopodium album L. for 24 h using flow cytometry after the cells were stained by propidium iodide. (D) Histograms present the percentage of each cell populations at the different cell cycle stage following treatment. Data presented as the mean ± standard deviation of at least three experiments. **P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Petroleum ether extract of Chenopodium album L. prevents cell growth and induces apoptosis of human lung cancer cells

    doi: 10.3892/etm.2016.3765

    Figure Lengend Snippet: Cell cycle analysis of A549 cells cultured with (A) 0.5% DMSO (control), and (B) 31.25 µg/ml and (C) 62.5 µg/ml of petroleum ether extract of Chenopodium album L. for 24 h using flow cytometry after the cells were stained by propidium iodide. (D) Histograms present the percentage of each cell populations at the different cell cycle stage following treatment. Data presented as the mean ± standard deviation of at least three experiments. **P

    Article Snippet: Finally, the cells were suspended in 500 µl staining buffer of 50 µg/ml propidium iodide (PI) with 100 µg/ml RNaseA (Beyotime Institute of Biotechnology), and incubated at 37°C for 30 min in the dark.

    Techniques: Cell Cycle Assay, Cell Culture, Flow Cytometry, Cytometry, Staining, Standard Deviation

    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

    In contrast to disruption of autophagy by p53-deficiency, interference with apoptosis either at the DISC or the mitochondrial level did not influence the autophagy process c-FLIP L , FADD-DN or Bcl-X L cDNA's were stably introduced into HCT116 wt cells by retroviral transduction and analyzed by western blotting and flow cytometry along with HCT116 p53 −/− cells and relevant controls. Non-transduced, empty vector-, FADD-DN-, c-FLIP L - and Bcl-X L -containing as well as HCT116 p53 −/− cells were harvested at the indicated time after 5-FU treatment and immuno-detection of cleaved PARP served as a marker of apoptosis, whereas p62 and LC3 staining were used as indicators of autophagy. Expression analyses of cFLIP L , FADD-DN and Bcl-X L were accomplished using specific antibodies. Probing of GAPDH was used to confirm equal loading of the samples. Cells were treated with 5-FU, either using 768 μM for 24 h A. or 10 μM for 72 h C. In parallel and using identical experimental conditions, general cell death was examined by propidium iodide labeling of fixed cells followed by flow cytometry analysis of SubG1 populations B. and D.

    Journal: Oncotarget

    Article Title: Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation

    doi: 10.18632/oncotarget.11073

    Figure Lengend Snippet: In contrast to disruption of autophagy by p53-deficiency, interference with apoptosis either at the DISC or the mitochondrial level did not influence the autophagy process c-FLIP L , FADD-DN or Bcl-X L cDNA's were stably introduced into HCT116 wt cells by retroviral transduction and analyzed by western blotting and flow cytometry along with HCT116 p53 −/− cells and relevant controls. Non-transduced, empty vector-, FADD-DN-, c-FLIP L - and Bcl-X L -containing as well as HCT116 p53 −/− cells were harvested at the indicated time after 5-FU treatment and immuno-detection of cleaved PARP served as a marker of apoptosis, whereas p62 and LC3 staining were used as indicators of autophagy. Expression analyses of cFLIP L , FADD-DN and Bcl-X L were accomplished using specific antibodies. Probing of GAPDH was used to confirm equal loading of the samples. Cells were treated with 5-FU, either using 768 μM for 24 h A. or 10 μM for 72 h C. In parallel and using identical experimental conditions, general cell death was examined by propidium iodide labeling of fixed cells followed by flow cytometry analysis of SubG1 populations B. and D.

    Article Snippet: Repeated washes in cold PBS and RNase A treatment (100 μg/ml, Invitrogen) for 1 h at 37°C were followed by propidium iodide staining (50 μg/ml, Sigma-Aldrich).

    Techniques: Stable Transfection, Transduction, Western Blot, Flow Cytometry, Cytometry, Plasmid Preparation, Marker, Staining, Expressing, Labeling

    Chloroquine and bafilomycin A abrogate 5-FU generated apoptosis in HCT116 cells through an effect separated from autophagy inhibition The effect of 5-FU with respect to the processing of caspases −8, −3 and PARP was analyzed by immunoblotting using total protein lysates from cells in which autophagy had been compromised by means of chloroquine (CQ, 20 μM), bafilomycin A (Baf A, 100 nM) or 3-methyladenine (3-MA, 5 mM) treatments. Detection of LC3 was performed to verify drug activities A. Cytotoxicity of 5-FU (768 μM, 20h), either used as a single agent or in combination with 10, 20 or 40 μM chloroquine (CQ), was examined by propidium iodide cell labeling followed by FACS analysis of SubG1 populations B. Along with siControl cells, siAtg5 C. siAtg7 D. siBeclin E. and siSQSTM1 (p62) F. transfected (20 nM) HCT116 wt cells were either left untreated or treated with 768 μM 5-FU for 24 h. Western blot examination of cell lysates with respect to cleaved lamin A (cle lamin A), cleaved PARP (cle PARP), p53 and siRNA efficiency are shown in (C–F). The apoptotic effects of 5-FU (768 μM), either alone or in co-treatments using cathepsin inhibitors E64d (5 μM) or pepstatin A (7 μM), or their combination, were analyzed by SDS-PAGE. Immuno-detection of DR5, p53, cleaved caspase-3 (cle casp-3) and LC3 in SDS-PAGE is outlined. G. Expression of DR5 and p53 in HCT116 wt cells as a result of CQ (10, 20, 40 or 80 μM) or 5-FU (768 μM) treatment, or their combinations was investigated by SDS-PAGE using HCT116 wt protein isolates. Immuno-detection of p62 and cleaved PARP served as markers for autophagy and apoptosis, respectively H. Along with controls, HCT116 wt and p5 3 −/− cells were treated with 5-FU (768 μM) or CQ (10 μM), or their combination. Isolated protein lysates were then separated by SDS-PAGE in order to analyze DR5, cleaved PARP, p53 and p62 using specific antibodies I. Immuno-detection of GAPDH or KU80 was used to control for equal loading of samples in (A and C–I). Processed caspase-8 fragments and the short isoform of DR5 are indicated by asterisks (A and G–I). An additional DR5-related fragment detected in western blot and appearing in CQ treated as well as in E64d and pepstatin A co-treated samples is indicated by an arrow (G–I).

    Journal: Oncotarget

    Article Title: Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation

    doi: 10.18632/oncotarget.11073

    Figure Lengend Snippet: Chloroquine and bafilomycin A abrogate 5-FU generated apoptosis in HCT116 cells through an effect separated from autophagy inhibition The effect of 5-FU with respect to the processing of caspases −8, −3 and PARP was analyzed by immunoblotting using total protein lysates from cells in which autophagy had been compromised by means of chloroquine (CQ, 20 μM), bafilomycin A (Baf A, 100 nM) or 3-methyladenine (3-MA, 5 mM) treatments. Detection of LC3 was performed to verify drug activities A. Cytotoxicity of 5-FU (768 μM, 20h), either used as a single agent or in combination with 10, 20 or 40 μM chloroquine (CQ), was examined by propidium iodide cell labeling followed by FACS analysis of SubG1 populations B. Along with siControl cells, siAtg5 C. siAtg7 D. siBeclin E. and siSQSTM1 (p62) F. transfected (20 nM) HCT116 wt cells were either left untreated or treated with 768 μM 5-FU for 24 h. Western blot examination of cell lysates with respect to cleaved lamin A (cle lamin A), cleaved PARP (cle PARP), p53 and siRNA efficiency are shown in (C–F). The apoptotic effects of 5-FU (768 μM), either alone or in co-treatments using cathepsin inhibitors E64d (5 μM) or pepstatin A (7 μM), or their combination, were analyzed by SDS-PAGE. Immuno-detection of DR5, p53, cleaved caspase-3 (cle casp-3) and LC3 in SDS-PAGE is outlined. G. Expression of DR5 and p53 in HCT116 wt cells as a result of CQ (10, 20, 40 or 80 μM) or 5-FU (768 μM) treatment, or their combinations was investigated by SDS-PAGE using HCT116 wt protein isolates. Immuno-detection of p62 and cleaved PARP served as markers for autophagy and apoptosis, respectively H. Along with controls, HCT116 wt and p5 3 −/− cells were treated with 5-FU (768 μM) or CQ (10 μM), or their combination. Isolated protein lysates were then separated by SDS-PAGE in order to analyze DR5, cleaved PARP, p53 and p62 using specific antibodies I. Immuno-detection of GAPDH or KU80 was used to control for equal loading of samples in (A and C–I). Processed caspase-8 fragments and the short isoform of DR5 are indicated by asterisks (A and G–I). An additional DR5-related fragment detected in western blot and appearing in CQ treated as well as in E64d and pepstatin A co-treated samples is indicated by an arrow (G–I).

    Article Snippet: Repeated washes in cold PBS and RNase A treatment (100 μg/ml, Invitrogen) for 1 h at 37°C were followed by propidium iodide staining (50 μg/ml, Sigma-Aldrich).

    Techniques: Generated, Inhibition, Labeling, FACS, Transfection, Western Blot, SDS Page, Expressing, Isolation

    Apoptosis by DAT1 upon blocking ERK activation a . Cells were treated with DAT1 in the presence or absence of the MEK inhibitor U0126. Subsequently, they were fixed and stained with DAPI and imaged in a fluorescence microscope. Percentage of apoptosis was calculated and plotted as given in Fig. 1 . *** denotes p ≤ 0.001 and indicates that the difference between the percentage of apoptosis caused by DAT1 in presence and absence of the inhibitor is extremely significant. b Cells were treated with DAT1 for 24 h with or without the presence of the MEK inhibitor and cell lysates were collected. Caspase 3 assay was performed according to manufacturer’s instructions and analysed as given in the methods section. ** for p ≤ 0.01 and indicates significant difference in the fluorescence intensities arising due to caspase 3 activation in the presence and absence of the inhibitor. c . ERK was inhibited by siRNA against ERK, followed by DAPI staining in the presence or absence of DAT1 and the percentage of apoptosis was calculated and plotted. d , e . Lack of DR5 activation upon blocking ERK activation was checked by western blotting. Indicated cell lines were treated with DAT1 for a period of 18 h in the presence or absence of the inhibitor ( d ) or in the presence or absence of ERK siRNA ( e )

    Journal: Molecular Cancer

    Article Title: ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells

    doi: 10.1186/s12943-016-0505-7

    Figure Lengend Snippet: Apoptosis by DAT1 upon blocking ERK activation a . Cells were treated with DAT1 in the presence or absence of the MEK inhibitor U0126. Subsequently, they were fixed and stained with DAPI and imaged in a fluorescence microscope. Percentage of apoptosis was calculated and plotted as given in Fig. 1 . *** denotes p ≤ 0.001 and indicates that the difference between the percentage of apoptosis caused by DAT1 in presence and absence of the inhibitor is extremely significant. b Cells were treated with DAT1 for 24 h with or without the presence of the MEK inhibitor and cell lysates were collected. Caspase 3 assay was performed according to manufacturer’s instructions and analysed as given in the methods section. ** for p ≤ 0.01 and indicates significant difference in the fluorescence intensities arising due to caspase 3 activation in the presence and absence of the inhibitor. c . ERK was inhibited by siRNA against ERK, followed by DAPI staining in the presence or absence of DAT1 and the percentage of apoptosis was calculated and plotted. d , e . Lack of DR5 activation upon blocking ERK activation was checked by western blotting. Indicated cell lines were treated with DAT1 for a period of 18 h in the presence or absence of the inhibitor ( d ) or in the presence or absence of ERK siRNA ( e )

    Article Snippet: Vinblastine, taxol, 5-Fluoro uracil, DAPI, propidium iodide and U0126 were from Sigma, USA. siRNA construct for ERK1/2 was obtained from Santacruz Biotechnology.

    Techniques: Blocking Assay, Activation Assay, Staining, Fluorescence, Microscopy, Caspase-3 Assay, Western Blot

    Neisseria gonorrhoeae induces cell death in monocyte-derived macrophages. MDMs were stimulated with medium alone (unstimulated), LPS (100 ng/mL, Escherichia coli O111:B4) and 5 mM ATP (LPS/ATP), 3 μM staurosporine (STS), formalin-fixed bacteria MOI 100 (FF MOI 100), or live bacteria MOI 10 or 100 of strain FA1090B. (A) Cell death was determined by trypan exclusion at 1, 6, 12, and 24 hours. (B) Cell death at 6 hours was confirmed by flow cytometry as the percent of total MDMs staining positive for propidium iodide. The graphs depict the mean ± SEM from 8 donors; 1-way ANOVA with a Dunnett multiple comparisons posttest was performed at each time point (* P

    Journal: Journal of Cell Death

    Article Title: Neisseria gonorrhoeae–Induced Inflammatory Pyroptosis in Human Macrophages is Dependent on Intracellular Gonococci and Lipooligosaccharide

    doi: 10.1177/1179066017750902

    Figure Lengend Snippet: Neisseria gonorrhoeae induces cell death in monocyte-derived macrophages. MDMs were stimulated with medium alone (unstimulated), LPS (100 ng/mL, Escherichia coli O111:B4) and 5 mM ATP (LPS/ATP), 3 μM staurosporine (STS), formalin-fixed bacteria MOI 100 (FF MOI 100), or live bacteria MOI 10 or 100 of strain FA1090B. (A) Cell death was determined by trypan exclusion at 1, 6, 12, and 24 hours. (B) Cell death at 6 hours was confirmed by flow cytometry as the percent of total MDMs staining positive for propidium iodide. The graphs depict the mean ± SEM from 8 donors; 1-way ANOVA with a Dunnett multiple comparisons posttest was performed at each time point (* P

    Article Snippet: Prior to analysis, cells were stained with the live-dead stain, propidium iodide (PI; BioLegend) and analyzed on a BD LSR II (San Jose, CA, USA).

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Staining

    Gating strategy for cell death detection with annexin and propidium iodide (PI) exemplified using untreated human osteoblasts. Analysis took place with FloJo. ( a ) Gating of cells from the debris using the side scatter height (SSC-H) and forward scatter height (FSC-H) channel. This gate was used to detect the different stainings in ( b ) where quadrant (Q)1 represents annexin single positive cells, Q2 annexin/PI double positive cells, Q3 PI single positive cells and Q4 annexin/PI double negative cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Devitalizing Effect of High Hydrostatic Pressure on Human Cells—Influence on Cell Death in Osteoblasts and Chondrocytes

    doi: 10.3390/ijms21113836

    Figure Lengend Snippet: Gating strategy for cell death detection with annexin and propidium iodide (PI) exemplified using untreated human osteoblasts. Analysis took place with FloJo. ( a ) Gating of cells from the debris using the side scatter height (SSC-H) and forward scatter height (FSC-H) channel. This gate was used to detect the different stainings in ( b ) where quadrant (Q)1 represents annexin single positive cells, Q2 annexin/PI double positive cells, Q3 PI single positive cells and Q4 annexin/PI double negative cells.

    Article Snippet: For precise analysis of cell death after HHP treatment, human osteoblasts and chondrocytes were analyzed by flow cytometry with the APC Annexin-V Apoptotis Detection Kit with propidium iodide (PI) (Biolegend, San Diego, CA, USA).

    Techniques:

    Analysis of cell death detection after HHP treatment (100–150 MPa and 250–300 MPa, 10 min) of ( a ) human osteoblast cell pellets ( n ≥ 4) and ( b ) human chondrocyte cell pellets ( n ≥ 5) with annexin and propidium iodide (PI). For each cell type, cell pellets were prepared for HHP treatment. Afterwards, treated cells were washed with autoMACS ® Running Buffer, stained with annexin and PI, and analyzed with the FACS Calibur. Data are shown as box plots. Significance between groups was detected via two-way ANOVA. ** p ≤ 0,01; **** p

    Journal: International Journal of Molecular Sciences

    Article Title: Devitalizing Effect of High Hydrostatic Pressure on Human Cells—Influence on Cell Death in Osteoblasts and Chondrocytes

    doi: 10.3390/ijms21113836

    Figure Lengend Snippet: Analysis of cell death detection after HHP treatment (100–150 MPa and 250–300 MPa, 10 min) of ( a ) human osteoblast cell pellets ( n ≥ 4) and ( b ) human chondrocyte cell pellets ( n ≥ 5) with annexin and propidium iodide (PI). For each cell type, cell pellets were prepared for HHP treatment. Afterwards, treated cells were washed with autoMACS ® Running Buffer, stained with annexin and PI, and analyzed with the FACS Calibur. Data are shown as box plots. Significance between groups was detected via two-way ANOVA. ** p ≤ 0,01; **** p

    Article Snippet: For precise analysis of cell death after HHP treatment, human osteoblasts and chondrocytes were analyzed by flow cytometry with the APC Annexin-V Apoptotis Detection Kit with propidium iodide (PI) (Biolegend, San Diego, CA, USA).

    Techniques: Staining, FACS

    Ca 2+ alleviation of H + and STOP1 . Seedlings grown for 5 d in a control solution (pH 5.5) were soaked in a solution containing 200 or 400 μ m CaCl 2 (pH 4.7) for 90 min and then stained with propidium iodide.

    Journal: Plant Physiology

    Article Title: Molecular and Physiological Analysis of Al3+ and H+ Rhizotoxicities at Moderately Acidic Conditions 1 Rhizotoxicities at Moderately Acidic Conditions 1 [W] Rhizotoxicities at Moderately Acidic Conditions 1 [W] [OPEN]

    doi: 10.1104/pp.113.222893

    Figure Lengend Snippet: Ca 2+ alleviation of H + and STOP1 . Seedlings grown for 5 d in a control solution (pH 5.5) were soaked in a solution containing 200 or 400 μ m CaCl 2 (pH 4.7) for 90 min and then stained with propidium iodide.

    Article Snippet: After 90 min of treatment, roots were stained with propidium iodide (3 μg mL–1 ) for 15 s and then observed under a fluorescent microscope (IMT-2-21-RFL; Olympus) using a dichroic mirror unit (IMT-2-DG; Olympus) and a density reduction filter (ND6; Olympus).

    Techniques: Staining

    Effects of indirubin and arsenic disulfide (As 2 S 2 ), alone or in combination, on the apoptosis of LY8 cells. LY8 cells were incubated with different doses of indirubin (1, 5, 10, 15 and 20 μM) and also 10 μM As 2 S 2 and 20 μM indirubin, alone or in combination, for 48 h. (A) The apoptotic rate was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining followed by flow cytometric analysis. The lower left quadrant (Q4) indicates the percentage of viable cells (Annexin V-FITC- and PI-negative), the upper left quadrant (Q1) indicates the percentage of early apoptotic cells (Annexin V-FITC-positive and PI-negative) and the upper right quadrant (Q2) indicates the percentage of late apoptotic cells (Annexin V-FITC- and PI-positive). (B) There were no significant effects on the rate of cell apoptosis of the LY8 cells following incubation with different doses of indirubin. However, significant differences were evident between the As 2 S 2 -treated group and the combination-treated group. Values are expressed as the mean ± standard deviation. ** P

    Journal: Oncology Letters

    Article Title: Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells

    doi: 10.3892/ol.2015.2941

    Figure Lengend Snippet: Effects of indirubin and arsenic disulfide (As 2 S 2 ), alone or in combination, on the apoptosis of LY8 cells. LY8 cells were incubated with different doses of indirubin (1, 5, 10, 15 and 20 μM) and also 10 μM As 2 S 2 and 20 μM indirubin, alone or in combination, for 48 h. (A) The apoptotic rate was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining followed by flow cytometric analysis. The lower left quadrant (Q4) indicates the percentage of viable cells (Annexin V-FITC- and PI-negative), the upper left quadrant (Q1) indicates the percentage of early apoptotic cells (Annexin V-FITC-positive and PI-negative) and the upper right quadrant (Q2) indicates the percentage of late apoptotic cells (Annexin V-FITC- and PI-positive). (B) There were no significant effects on the rate of cell apoptosis of the LY8 cells following incubation with different doses of indirubin. However, significant differences were evident between the As 2 S 2 -treated group and the combination-treated group. Values are expressed as the mean ± standard deviation. ** P

    Article Snippet: Assessment of apoptosis using Annexin V and propidium iodide (PI) The apoptotic rate of the LY1 and LY8 cells was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGen Biotech Co., Ltd., Nanjing, China).

    Techniques: Incubation, Staining, Flow Cytometry, Standard Deviation

    Effects of indirubin and arsenic disulfide (As 2 S 2 ), alone or in combination, on the apoptosis of LY1 cells. LY1 cells were incubated with different doses of indirubin (1, 5, 10, 15 and 20 μM) and also 10 μM As 2 S 2 and 20 μM indirubin, alone or in combination, for 48 h. (A) The apoptotic rate was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining followed by flow cytometric analysis. The lower left quadrant (Q4) indicates the percentage of viable cells (Annexin V-FITC- and PI-negative), the upper left quadrant (Q1) indicates the percentage of early apoptotic cells (Annexin V-FITC-positive and PI-negative) and the upper right quadrant (Q2) indicates the percentage of late apoptotic cells (Annexin V-FITC- and PI-positive). (B) There were no significant effects on the rate of apoptosis of the LY1 cells following incubation with different doses of indirubin. However, significant differences were evident between the As 2 S 2 -treated group and the combination-treated group. Values are expressed as the mean ± standard deviation. ** P

    Journal: Oncology Letters

    Article Title: Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells

    doi: 10.3892/ol.2015.2941

    Figure Lengend Snippet: Effects of indirubin and arsenic disulfide (As 2 S 2 ), alone or in combination, on the apoptosis of LY1 cells. LY1 cells were incubated with different doses of indirubin (1, 5, 10, 15 and 20 μM) and also 10 μM As 2 S 2 and 20 μM indirubin, alone or in combination, for 48 h. (A) The apoptotic rate was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining followed by flow cytometric analysis. The lower left quadrant (Q4) indicates the percentage of viable cells (Annexin V-FITC- and PI-negative), the upper left quadrant (Q1) indicates the percentage of early apoptotic cells (Annexin V-FITC-positive and PI-negative) and the upper right quadrant (Q2) indicates the percentage of late apoptotic cells (Annexin V-FITC- and PI-positive). (B) There were no significant effects on the rate of apoptosis of the LY1 cells following incubation with different doses of indirubin. However, significant differences were evident between the As 2 S 2 -treated group and the combination-treated group. Values are expressed as the mean ± standard deviation. ** P

    Article Snippet: Assessment of apoptosis using Annexin V and propidium iodide (PI) The apoptotic rate of the LY1 and LY8 cells was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGen Biotech Co., Ltd., Nanjing, China).

    Techniques: Incubation, Staining, Flow Cytometry, Standard Deviation

    Cell cycle analysis. GL15 (A) , U87MG (B) and U251 (C) cells were treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin for 24 h and 48 h respectively. Cell distribution in G0/G1, S and G2/M phases was analyzed by flow cytometry using propidium iodide DNA staining. Legend: *Any inhibitor/control, # PP242/wortmannin, ≈ PP242/rapamycin, ∧ rapamycin/wortmannin ( *,#,∧,≈ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: PP242 Counteracts Glioblastoma Cell Proliferation, Migration, Invasiveness and Stemness Properties by Inhibiting mTORC2/AKT

    doi: 10.3389/fncel.2018.00099

    Figure Lengend Snippet: Cell cycle analysis. GL15 (A) , U87MG (B) and U251 (C) cells were treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin for 24 h and 48 h respectively. Cell distribution in G0/G1, S and G2/M phases was analyzed by flow cytometry using propidium iodide DNA staining. Legend: *Any inhibitor/control, # PP242/wortmannin, ≈ PP242/rapamycin, ∧ rapamycin/wortmannin ( *,#,∧,≈ p

    Article Snippet: Attached cells were washed twice with PBS and resuspended with 0.5 ml of hypotonic propidium iodide solution (50 μg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) in 12 × 75 mm polypropylene tubes (BD Biosciences).

    Techniques: Cell Cycle Assay, Flow Cytometry, Cytometry, Staining

    NCS‐01 cells, in part, employ filopodia‐mediated long distance rescue of cultured primary rat cortical cells against oxygen glucose deprivation (OGD). Primary rat cortical cells were subjected to OGD and reperfusion then cocultured with NCS‐01 cells at different distances, as depicted by procedural timeline and diagram (A and A′). Propidium iodide and N‐cadherin staining was conducted for the different distances: (B and B′) 1.68 mm, (C and C′) 1.80 mm, (D and D′) 1.92 mm, and (E and E′) 2.04 mm. Propidium iodide staining (red) reveals fewer dead primary rat cortical cells (indicated by red color) at closer distances than at farther distances between upper and lower chambers (B‐E). N‐cadherin staining (green) indicates the presence of filopodia extending from NCS‐01 cells (B′‐E′) Scale bar = 50 μm. Quantitative analysis of primary rat cortical neurons reveals that cell viability correlates inversely with increased distance between NCS‐01 cells and primary rat cortical neurons, when measured by trypan blue (TB) assay (F). Similarly, MTT assay reveals that greater mitochondrial activity correlates inversely with greater distance between NCS‐01 cells and primary rat cortical neurons (G). Significance depicted as a‐d at P

    Journal: Stem Cells Translational Medicine

    Article Title: Translating intracarotid artery transplantation of bone marrow‐derived NCS‐01 cells for ischemic stroke: Behavioral and histological readouts and mechanistic insights into stem cell therapy, et al. Translating intracarotid artery transplantation of bone marrow‐derived NCS‐01 cells for ischemic stroke: Behavioral and histological readouts and mechanistic insights into stem cell therapy

    doi: 10.1002/sctm.19-0229

    Figure Lengend Snippet: NCS‐01 cells, in part, employ filopodia‐mediated long distance rescue of cultured primary rat cortical cells against oxygen glucose deprivation (OGD). Primary rat cortical cells were subjected to OGD and reperfusion then cocultured with NCS‐01 cells at different distances, as depicted by procedural timeline and diagram (A and A′). Propidium iodide and N‐cadherin staining was conducted for the different distances: (B and B′) 1.68 mm, (C and C′) 1.80 mm, (D and D′) 1.92 mm, and (E and E′) 2.04 mm. Propidium iodide staining (red) reveals fewer dead primary rat cortical cells (indicated by red color) at closer distances than at farther distances between upper and lower chambers (B‐E). N‐cadherin staining (green) indicates the presence of filopodia extending from NCS‐01 cells (B′‐E′) Scale bar = 50 μm. Quantitative analysis of primary rat cortical neurons reveals that cell viability correlates inversely with increased distance between NCS‐01 cells and primary rat cortical neurons, when measured by trypan blue (TB) assay (F). Similarly, MTT assay reveals that greater mitochondrial activity correlates inversely with greater distance between NCS‐01 cells and primary rat cortical neurons (G). Significance depicted as a‐d at P

    Article Snippet: Cultures were stained with propidium iodide to reveal cell viability and N‐cadherin to detect filopodia (Figure B‐E').

    Techniques: Cell Culture, Staining, MTT Assay, Activity Assay