propidium iodide Search Results


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  • 99
    Thermo Fisher propidium iodide
    Knock-down of endogenous Ginir affects proliferation and tumourigenic potential of fibroblasts and melanoma cells. (A) Representative RT-PCR for analyses of Ginir knock-down in NIH/3T3 cells expressing Ginir-shRNA (1, 2, and scrambled control) using G2F-G2R primers. Gapdh served as an internal control. (B) Cell proliferation analysis by MTT assay for the indicated cell lines performed over a period of 4 days. Data presented are mean ± SEM. *** P ≤ 0.0001, one tailed, by two-way ANOVA test. (C) Quantification of Ki67 antigen–expressing cells shown as percentage of positively stained cells from the total number of cells present per field. Cells from at least 10 representative fields were counted in NIH/3T3 control and Ginir shRNA1 and 2 cells. Data presented are mean ± SEM.** P ≤ 0.001 by one-way ANOVA test ( n = 3). (D) Quantitative analyses of cell cycle parameters of PI-stained cells in flow cytometry. Stable Ginir knock-down (NIHshGinir1, NIHshGinir2) cells were compared to NIH/3T3 control cells having endogenous Ginir RNA expression for percentage of cells in various phases of the cell cycle. Bars: means ± SEM; * P ≤ 0.05, ** P ≤ 0.001; two tailed; by two-way ANOVA test. (E) Representative RT-PCR data demonstrating Ginir RNA expression levels in B16F10 melanoma cells shControl and the knock-down cells B16F10-Ginir-shRNA1 and B16F10-GinirshRNA2. PCR was done using G2F-G2R primers. Gapdh RNA expression was used for normalisation. (F) Phase contrast images of B16F10 control cells and cells stably transfected with shGinir1 or shGinir2. Arrowheads point towards cells showing altered phenotypes. Magnification 10×. (G) Cell proliferation analysis of indicated cells performed by MTT assay over a period of 4 days. Data shown are mean ± SEM. * P ≤ 0.05, one tailed, Student’s paired t test ( n = 3). (H) Quantification of Ki67 antigen expression shown as percentage of positively stained cells as compared to the total number of cells per field. At least 10 fields were counted. Data shown are mean ± SEM. ** P ≤ 0.001 by one-way ANOVA test. ( n = 3). (I) Cell migration assay in the indicated cell lines. The gaps were measured after 6 hours using ImageJ software; version 1.41. Experiment was repeated at least thrice. (J) Quantitative analysis of relative wound recovery of each B16F10 Ginir knock-down cell induced by shRNA1 and shRNA2 as compared to B16F10-shGinir scrambled expressing control cells. Data represent mean ± SEM ( n = 3). *** P ≤ 0.0001 by two-way ANOVA test. (K) Representative xenograft tumours of B16F10-shControl and B16F10-shGinir1 and B16F10shGinir2 (1 and 2) cells introduced into NOD/SCID mice; tumour growth was monitored for 15 days. These experiments were performed thrice, with 3 mice in each group. (L) Tumour growth kinetics were determined by measuring tumour volume each day for a period of 15 days for the indicated cell lines. Data shown represent mean ± SEM. *** P . Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; PI, <t>propidium</t> iodide; RT-PCR, reverse transcription PCR; shRNA, short hairpin RNA.
    Propidium Iodide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42022 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore propidium iodide
    Reduced cell proliferation and increased apoptosis appear in Tnks DKO crypts. (A, B) Immunofluorescence analysis of small intestinal (A) or colon (B) sections from mice at day 4 after the first TAM injection shows the expression of the proliferation marker Ki67. Left panel: Representative images from five mice of each genotype are depicted. Scale bar: 80 μm (A) and 100 μm (B). Right panel: Quantification of the Ki67 positive cells in crypts (n = 4 mice). (C) Small intestine crypt cells were isolated from control and Tnks DKO mice at day 4 after the first TAM injection, stained by <t>propidium</t> iodide (PI), and then subjected to FACS analysis. Left panel: A representative FACS result from 5 mice of each genotype is depicted. Right panel: Quantification of the percentage of dead cells (n = 6 mice). (D) TUNEL assay of small intestine of mice at day 4 after the first TAM injection. Left panel: Representative images from 4 mice of each genotype are depicted. Cell nuclei were counterstained with <t>DAPI.</t> Scale bar: 50 μm. Right panel: Quantification of apoptotic cells of the indicated adult mice (n = 4). 40 fields were scored for each condition. Data are represented as means ± SD, analyzed by two-way ANOVA test. *P
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    92
    Becton Dickinson propidium iodide
    The IRE1 pathway is not necessary for MMC-induced JNK activation. (A) JNK activation in response to MMC treatment (0.4 mg/ml, 5 minutes) was detected at various times by Western blotting. (B) Fibroblasts were pretreated with a 5 µM concentration of the JNK inhibitor SP600125, then treated with MMC (0.4 mg/ml, 5 minutes). The expression of P-JNK, JNK, and β-actin (loading control) was measured by Western blotting 24 hours after MMC treatment. (C) Cell viability and (D) apoptosis rates were quantified using CCK-8 kit assays and Annexin <t>V/propidium</t> iodide double staining, 48 hours after MMC treatment. The histograms represent the mean ± SD of three independent experiments. **P
    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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    Becton Dickinson propidium iodide pi
    Phosphatidylinositol 3-kinase/Akt acts downstream of FAK signaling to regulate apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with LY294002. (A and B) The expression of Akt, pAkt, caspase-3 and c-caspase-3 was examined using western blotting. (C and D) Cell apoptosis was examined using (C) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (D) Annexin <t>V/propidium</t> iodide. (E and F) The expression of FAK and pFAK was examined using western blotting. Scale bar, 200 µm. FAK, focal adhesion kinase; pFAK, phosphorylated FAK; c-caspase-3, cleaved caspase-3; TGFβ, transforming growth factor-β.
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    Millipore propidium iodide solution
    Neutralization of the PM-MSC complex . (a, b) Viability of rat fetal membrane-derived MSCs at 10 min after mixture of PM (final dose 0.8%) with or without neutralization using NaOH in-vitro was assessed by <t>propidium</t> iodide staining. Representative pictures in each group are presented (a). Scale bar = 50 μm. The bar graph presents the averaged data (b). n=4 in each point ; * p
    Propidium Iodide Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2085 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beyotime propidium iodide
    Cell apoptosis analysis in HEPG2 and MHCC97-H liver cancer cells using Annexin V-FITC/PI staining and flow cytometry. (A) Cell apoptosis analysis by flow cytometry. Early-stage apoptotic cells are presented in the lower right quadrants (Annexin V-FITC positive and PI negative) and late-stage apoptotic cells are presented in the upper right quadrants (Annexin V-FITC and PI positive) (B) Calculated apoptotic rate (%) of early and late stage apoptotic cells. Similar results were obtained in three independent experiments. Results are presented as the mean ± standard error of the mean. FITC, fluorescein isothiocyanate; PI, <t>propidium</t> iodide; siRNA, small interfering RNA.
    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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    Becton Dickinson propidium iodide staining
    PCB-induced arrest is reversible and cells regain sensitivity to VCR after reentering the cell cycle. A . ALL-5 and T98G cells were stained with <t>propidium</t> iodide and subjected to two-parameter flow cytometry to create a gate distinguishing EdU-negative and EdU-positive cells (left panels). ALL-5 and T98G cells were then treated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), washed thrice with PBS, and incubated in growth media containing 10 µM EdU for the times indicated. Cells were harvested, fixed, and stained for EdU incorporation and with propidium iodide and analyzed by two-parameter flow cytometry. Data shown are representative of three independent experiments. The percentage of cells positive for EdU is indicated at the top of each panel. B . ALL-5 and T98G cells were treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h with or without pretreatment with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G). A third set of cells were pretreated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), after which cells were washed thrice with PBS and treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h. Cells were stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with
    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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    93
    Beyotime propidium iodide pi
    Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by <t>propidium</t> iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P
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    Thermo Fisher propidium iodide 1 0 solution in water
    Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by <t>propidium</t> iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P
    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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    99
    BioLegend propidium iodide
    Baricitinib suppresses CD80/CD86 expression on dendritic cells (DCs). Immature monocyte-derived dendritic cells were cultured with or without baricitinib and tofacitinib during lipopolysaccharide stimulation for 48 h. The DC phenotype was evaluated using flow cytometry. (A) Expression of HLA-DR, CD80, and CD86. (B) Representative histogram data of HLA-DR, CD80, and CD86 expression. (C) Rate of viable cells (annexin V neg <t>/propidium</t> iodide neg ). (D) Expression of CD80 and CD86 in the presence of baricitinib and tofacitinib. Data are mean ± SD of three different donors per group. * 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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    Keygen Biotech propidium iodide
    Radioresistant TE-1 and Eca-109 cells showed more aggressive malignancies. (A) Wound-healing assay of TE-1 and TE-1/R cells. (B) Wound-healing assay of Eca-109 and Eca-109/R cells. Wound healing was observed 24 h after the treatment. (C) Induction of apoptosis by radiation in TE-1 and TE-1/R cells. (D) Induction of apoptosis by radiation in Eca-109 and Eca-109/R cells. Cells were treated with 6 Gy irradiation, and the apoptosis was measured using <t>propidium</t> iodide (PI)/Annexin-V double staining. Data are normalized to the control cells and presented as the mean ± SEM of three independent experiments, * P
    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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    92
    Carl Zeiss propidium iodide
    Confocal microscopy of MG-63 cells seeded on ST1 and ST2 - day 14. Confocal microscopy of MG-63 cells seeded on ST1 ( a , c ) or ST2 ( b , d ) scaffolds from polylactic acid after a 14-day culture. Cells were fixed and cell membranes were stained using DiOC6 (3) ( green ), cell nuclei were stained with <t>propidium</t> iodide ( red ). Both maximum projections ( a - b ) and color coded projections ( c , d ), which display depth ( d ) distribution of cells (d = 180 μm in C, d = 200 μm in D) showed confluent layer of MG-63 cells and formation of bridges from cells connecting fibres on both scaffolds. Objective ×10, magnification ×2, Bar = 50 μm
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    Olympus propidium iodide
    Effect of combined RQC or individual quercetin on breast cancer cell proliferation. MDA-MB-231 and MDA-MB-435 cells in 5% serum and phenol red-free media were treated with vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each or 15μM quercetin for 48h. Cells were fixed, nuclei stained with <t>Propidium</t> Iodide (PI), and intact (non-apoptotic) nuclei quantified. Percentage of viable cells ± SEM for 30 microscopic fields/triplicate treatments (N = 3) is presented. A) Average cell viability of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. (B) Average cell viability of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.
    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
    The effect of oridonin nanosuspension and free oridonin on PANC-1 cell apoptosis was measured by annexin V-fluorescein <t>isothiocyanate/propidium</t> iodide staining. The early apoptotic cells stained by annexin-V-fluorescein isothiocyanate are located in the lower right quadrant. Abbreviation: ORI, oridonin.
    Propidium Iodide Pi, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 92/100, based on 690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson propidium iodide solution
    SB225002 induces cell death in ALL cell lines. Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of (A) B-ALL and T-ALL cell lines. (B) Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. (C) Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. (D) Annexin-V and <t>propidium</t> iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.
    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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    N/A
    Propidium iodide PI is membrane impermeant and generally excluded from viable cells However it can easily penetrate dead or damaged cells and as such is commonly used for identifying cell
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    N/A
    Propidium Iodide PI binds to double stranded DNA and RNA after cells have been permeabilized Once bound to nucleic acids the nuclei appear red in color as observed by fluorescence
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    Knock-down of endogenous Ginir affects proliferation and tumourigenic potential of fibroblasts and melanoma cells. (A) Representative RT-PCR for analyses of Ginir knock-down in NIH/3T3 cells expressing Ginir-shRNA (1, 2, and scrambled control) using G2F-G2R primers. Gapdh served as an internal control. (B) Cell proliferation analysis by MTT assay for the indicated cell lines performed over a period of 4 days. Data presented are mean ± SEM. *** P ≤ 0.0001, one tailed, by two-way ANOVA test. (C) Quantification of Ki67 antigen–expressing cells shown as percentage of positively stained cells from the total number of cells present per field. Cells from at least 10 representative fields were counted in NIH/3T3 control and Ginir shRNA1 and 2 cells. Data presented are mean ± SEM.** P ≤ 0.001 by one-way ANOVA test ( n = 3). (D) Quantitative analyses of cell cycle parameters of PI-stained cells in flow cytometry. Stable Ginir knock-down (NIHshGinir1, NIHshGinir2) cells were compared to NIH/3T3 control cells having endogenous Ginir RNA expression for percentage of cells in various phases of the cell cycle. Bars: means ± SEM; * P ≤ 0.05, ** P ≤ 0.001; two tailed; by two-way ANOVA test. (E) Representative RT-PCR data demonstrating Ginir RNA expression levels in B16F10 melanoma cells shControl and the knock-down cells B16F10-Ginir-shRNA1 and B16F10-GinirshRNA2. PCR was done using G2F-G2R primers. Gapdh RNA expression was used for normalisation. (F) Phase contrast images of B16F10 control cells and cells stably transfected with shGinir1 or shGinir2. Arrowheads point towards cells showing altered phenotypes. Magnification 10×. (G) Cell proliferation analysis of indicated cells performed by MTT assay over a period of 4 days. Data shown are mean ± SEM. * P ≤ 0.05, one tailed, Student’s paired t test ( n = 3). (H) Quantification of Ki67 antigen expression shown as percentage of positively stained cells as compared to the total number of cells per field. At least 10 fields were counted. Data shown are mean ± SEM. ** P ≤ 0.001 by one-way ANOVA test. ( n = 3). (I) Cell migration assay in the indicated cell lines. The gaps were measured after 6 hours using ImageJ software; version 1.41. Experiment was repeated at least thrice. (J) Quantitative analysis of relative wound recovery of each B16F10 Ginir knock-down cell induced by shRNA1 and shRNA2 as compared to B16F10-shGinir scrambled expressing control cells. Data represent mean ± SEM ( n = 3). *** P ≤ 0.0001 by two-way ANOVA test. (K) Representative xenograft tumours of B16F10-shControl and B16F10-shGinir1 and B16F10shGinir2 (1 and 2) cells introduced into NOD/SCID mice; tumour growth was monitored for 15 days. These experiments were performed thrice, with 3 mice in each group. (L) Tumour growth kinetics were determined by measuring tumour volume each day for a period of 15 days for the indicated cell lines. Data shown represent mean ± SEM. *** P . Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; PI, propidium iodide; RT-PCR, reverse transcription PCR; shRNA, short hairpin RNA.

    Journal: PLoS Biology

    Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

    doi: 10.1371/journal.pbio.2004204

    Figure Lengend Snippet: Knock-down of endogenous Ginir affects proliferation and tumourigenic potential of fibroblasts and melanoma cells. (A) Representative RT-PCR for analyses of Ginir knock-down in NIH/3T3 cells expressing Ginir-shRNA (1, 2, and scrambled control) using G2F-G2R primers. Gapdh served as an internal control. (B) Cell proliferation analysis by MTT assay for the indicated cell lines performed over a period of 4 days. Data presented are mean ± SEM. *** P ≤ 0.0001, one tailed, by two-way ANOVA test. (C) Quantification of Ki67 antigen–expressing cells shown as percentage of positively stained cells from the total number of cells present per field. Cells from at least 10 representative fields were counted in NIH/3T3 control and Ginir shRNA1 and 2 cells. Data presented are mean ± SEM.** P ≤ 0.001 by one-way ANOVA test ( n = 3). (D) Quantitative analyses of cell cycle parameters of PI-stained cells in flow cytometry. Stable Ginir knock-down (NIHshGinir1, NIHshGinir2) cells were compared to NIH/3T3 control cells having endogenous Ginir RNA expression for percentage of cells in various phases of the cell cycle. Bars: means ± SEM; * P ≤ 0.05, ** P ≤ 0.001; two tailed; by two-way ANOVA test. (E) Representative RT-PCR data demonstrating Ginir RNA expression levels in B16F10 melanoma cells shControl and the knock-down cells B16F10-Ginir-shRNA1 and B16F10-GinirshRNA2. PCR was done using G2F-G2R primers. Gapdh RNA expression was used for normalisation. (F) Phase contrast images of B16F10 control cells and cells stably transfected with shGinir1 or shGinir2. Arrowheads point towards cells showing altered phenotypes. Magnification 10×. (G) Cell proliferation analysis of indicated cells performed by MTT assay over a period of 4 days. Data shown are mean ± SEM. * P ≤ 0.05, one tailed, Student’s paired t test ( n = 3). (H) Quantification of Ki67 antigen expression shown as percentage of positively stained cells as compared to the total number of cells per field. At least 10 fields were counted. Data shown are mean ± SEM. ** P ≤ 0.001 by one-way ANOVA test. ( n = 3). (I) Cell migration assay in the indicated cell lines. The gaps were measured after 6 hours using ImageJ software; version 1.41. Experiment was repeated at least thrice. (J) Quantitative analysis of relative wound recovery of each B16F10 Ginir knock-down cell induced by shRNA1 and shRNA2 as compared to B16F10-shGinir scrambled expressing control cells. Data represent mean ± SEM ( n = 3). *** P ≤ 0.0001 by two-way ANOVA test. (K) Representative xenograft tumours of B16F10-shControl and B16F10-shGinir1 and B16F10shGinir2 (1 and 2) cells introduced into NOD/SCID mice; tumour growth was monitored for 15 days. These experiments were performed thrice, with 3 mice in each group. (L) Tumour growth kinetics were determined by measuring tumour volume each day for a period of 15 days for the indicated cell lines. Data shown represent mean ± SEM. *** P . Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; PI, propidium iodide; RT-PCR, reverse transcription PCR; shRNA, short hairpin RNA.

    Article Snippet: Nuclei were stained with PI (Invitrogen, # P1304MP) for 30 minutes.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA, MTT Assay, One-tailed Test, Staining, Flow Cytometry, Cytometry, RNA Expression, Two Tailed Test, Polymerase Chain Reaction, Stable Transfection, Transfection, Cell Migration Assay, Software, Mouse Assay

    Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Journal: Stem Cells International

    Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    doi: 10.1155/2018/5034679

    Figure Lengend Snippet: Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Article Snippet: Cells were counted after fixation and propidium iodide staining by TALI® cytometry (Life Technologies).

    Techniques: Incubation, Immunofluorescence, Expressing, Marker, Fluorescence, Western Blot, SDS Page

    Reduced cell proliferation and increased apoptosis appear in Tnks DKO crypts. (A, B) Immunofluorescence analysis of small intestinal (A) or colon (B) sections from mice at day 4 after the first TAM injection shows the expression of the proliferation marker Ki67. Left panel: Representative images from five mice of each genotype are depicted. Scale bar: 80 μm (A) and 100 μm (B). Right panel: Quantification of the Ki67 positive cells in crypts (n = 4 mice). (C) Small intestine crypt cells were isolated from control and Tnks DKO mice at day 4 after the first TAM injection, stained by propidium iodide (PI), and then subjected to FACS analysis. Left panel: A representative FACS result from 5 mice of each genotype is depicted. Right panel: Quantification of the percentage of dead cells (n = 6 mice). (D) TUNEL assay of small intestine of mice at day 4 after the first TAM injection. Left panel: Representative images from 4 mice of each genotype are depicted. Cell nuclei were counterstained with DAPI. Scale bar: 50 μm. Right panel: Quantification of apoptotic cells of the indicated adult mice (n = 4). 40 fields were scored for each condition. Data are represented as means ± SD, analyzed by two-way ANOVA test. *P

    Journal: PLoS Genetics

    Article Title: Tankyrases maintain homeostasis of intestinal epithelium by preventing cell death

    doi: 10.1371/journal.pgen.1007697

    Figure Lengend Snippet: Reduced cell proliferation and increased apoptosis appear in Tnks DKO crypts. (A, B) Immunofluorescence analysis of small intestinal (A) or colon (B) sections from mice at day 4 after the first TAM injection shows the expression of the proliferation marker Ki67. Left panel: Representative images from five mice of each genotype are depicted. Scale bar: 80 μm (A) and 100 μm (B). Right panel: Quantification of the Ki67 positive cells in crypts (n = 4 mice). (C) Small intestine crypt cells were isolated from control and Tnks DKO mice at day 4 after the first TAM injection, stained by propidium iodide (PI), and then subjected to FACS analysis. Left panel: A representative FACS result from 5 mice of each genotype is depicted. Right panel: Quantification of the percentage of dead cells (n = 6 mice). (D) TUNEL assay of small intestine of mice at day 4 after the first TAM injection. Left panel: Representative images from 4 mice of each genotype are depicted. Cell nuclei were counterstained with DAPI. Scale bar: 50 μm. Right panel: Quantification of apoptotic cells of the indicated adult mice (n = 4). 40 fields were scored for each condition. Data are represented as means ± SD, analyzed by two-way ANOVA test. *P

    Article Snippet: N-acetylcysteine, BSA, DAPI, EDTA and Propidium Iodide were from Sigma.

    Techniques: Immunofluorescence, Mouse Assay, Injection, Expressing, Marker, Isolation, Staining, FACS, TUNEL Assay

    The IRE1 pathway is not necessary for MMC-induced JNK activation. (A) JNK activation in response to MMC treatment (0.4 mg/ml, 5 minutes) was detected at various times by Western blotting. (B) Fibroblasts were pretreated with a 5 µM concentration of the JNK inhibitor SP600125, then treated with MMC (0.4 mg/ml, 5 minutes). The expression of P-JNK, JNK, and β-actin (loading control) was measured by Western blotting 24 hours after MMC treatment. (C) Cell viability and (D) apoptosis rates were quantified using CCK-8 kit assays and Annexin V/propidium iodide double staining, 48 hours after MMC treatment. The histograms represent the mean ± SD of three independent experiments. **P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    doi: 10.1371/journal.pone.0059330

    Figure Lengend Snippet: The IRE1 pathway is not necessary for MMC-induced JNK activation. (A) JNK activation in response to MMC treatment (0.4 mg/ml, 5 minutes) was detected at various times by Western blotting. (B) Fibroblasts were pretreated with a 5 µM concentration of the JNK inhibitor SP600125, then treated with MMC (0.4 mg/ml, 5 minutes). The expression of P-JNK, JNK, and β-actin (loading control) was measured by Western blotting 24 hours after MMC treatment. (C) Cell viability and (D) apoptosis rates were quantified using CCK-8 kit assays and Annexin V/propidium iodide double staining, 48 hours after MMC treatment. The histograms represent the mean ± SD of three independent experiments. **P

    Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.

    Techniques: Activation Assay, Western Blot, Concentration Assay, Expressing, CCK-8 Assay, Double Staining

    Application of MMC for 5 minutes induces apoptosis in human fibroblasts. (A) Cell viability after different treatments was detected by using the CCK-8 assay. MMC-induced growth inhibition occurred in a dose- and time-dependent manner. (B) Apoptosis analysis was assessed via Annexin V/propidium iodide double staining. Cells were exposed to 0.4 mg/ml MMC for 5 minutes and then incubated for the corresponding indicated times. Apoptosis rates were determined via flow cytometry analysis. The cells shown in the bottom right quadrant were Annexin V-FITC positive and propidium iodide negative, indicating an early stage of apoptosis. The cells in the top right quadrant stained positively for Annexin V-FITC and propidium iodide, indicating that they consisted of secondary late apoptotic/necrotic cells. The total percentage of cell death is shown in bold. Statistical analysis of the total recorded apoptotic cells was performed, and the results are shown in the bar graphs. (C) Cell cycle analysis was conducted to detect the changes in the cell cycle at different time points after MMC treatment (0.4 mg/ml, 5 minutes). (D) Cells were TUNEL stained 48 hours after MMC treatment and then observed using a fluorescence microscope. Nuclei are shown in blue, and TUNEL staining is shown in green. (E) Western blots revealed that MMC induces cleavage of caspase-3 from a size of 35 kDa to 17 kDa and the degradation of poly ADP-ribose polymerase (PARP) by the cleaved caspase-3 into 85 to 90 kDa fragments. β-actin was included as a control. Gels were run in triplicate. The histograms in panels A, B, C and D represent the mean ±SD of three independent experiments. *P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    doi: 10.1371/journal.pone.0059330

    Figure Lengend Snippet: Application of MMC for 5 minutes induces apoptosis in human fibroblasts. (A) Cell viability after different treatments was detected by using the CCK-8 assay. MMC-induced growth inhibition occurred in a dose- and time-dependent manner. (B) Apoptosis analysis was assessed via Annexin V/propidium iodide double staining. Cells were exposed to 0.4 mg/ml MMC for 5 minutes and then incubated for the corresponding indicated times. Apoptosis rates were determined via flow cytometry analysis. The cells shown in the bottom right quadrant were Annexin V-FITC positive and propidium iodide negative, indicating an early stage of apoptosis. The cells in the top right quadrant stained positively for Annexin V-FITC and propidium iodide, indicating that they consisted of secondary late apoptotic/necrotic cells. The total percentage of cell death is shown in bold. Statistical analysis of the total recorded apoptotic cells was performed, and the results are shown in the bar graphs. (C) Cell cycle analysis was conducted to detect the changes in the cell cycle at different time points after MMC treatment (0.4 mg/ml, 5 minutes). (D) Cells were TUNEL stained 48 hours after MMC treatment and then observed using a fluorescence microscope. Nuclei are shown in blue, and TUNEL staining is shown in green. (E) Western blots revealed that MMC induces cleavage of caspase-3 from a size of 35 kDa to 17 kDa and the degradation of poly ADP-ribose polymerase (PARP) by the cleaved caspase-3 into 85 to 90 kDa fragments. β-actin was included as a control. Gels were run in triplicate. The histograms in panels A, B, C and D represent the mean ±SD of three independent experiments. *P

    Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.

    Techniques: CCK-8 Assay, Inhibition, Double Staining, Incubation, Flow Cytometry, Cytometry, Staining, Cell Cycle Assay, TUNEL Assay, Fluorescence, Microscopy, Western Blot

    Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants. (A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    doi: 10.1371/journal.pone.0059330

    Figure Lengend Snippet: Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants. (A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P

    Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.

    Techniques: Fluorescence, Microscopy, Staining, Incubation, Multiple Displacement Amplification, Flow Cytometry, Cytometry, CCK-8 Assay, Double Staining, Western Blot, Expressing

    CHOP is essential for MMC-induced apoptosis. Fibroblasts were transfected with CHOP or control siRNA (non-targeting siRNA). After treatment with 0.4 mg/ml MMC for 48 hours, the cells were analyzed in a number of assays. (A) Cell viability was assessed with the CCK-8 assay. (B) Annexin V/propidium iodide double staining was performed to detect the apoptosis rate. (C) TUNEL-stained cells were observed under a fluorescence microscope. (D) Whole-cell lysates were used for Western blotting with antibodies specific for CHOP, BAX, BCL-2, BIM and β-actin (loading control). This experiment was performed in triplicate. (E) The band intensities for CHOP, BCL-2/BAX and BIM were expressed as a histogram relative to β-actin. The control group (no MMC treatment) was normalized to a value of 1.0-fold. The data in panels A, B, C and E are the mean ± SD of at least three independent experiments. *P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    doi: 10.1371/journal.pone.0059330

    Figure Lengend Snippet: CHOP is essential for MMC-induced apoptosis. Fibroblasts were transfected with CHOP or control siRNA (non-targeting siRNA). After treatment with 0.4 mg/ml MMC for 48 hours, the cells were analyzed in a number of assays. (A) Cell viability was assessed with the CCK-8 assay. (B) Annexin V/propidium iodide double staining was performed to detect the apoptosis rate. (C) TUNEL-stained cells were observed under a fluorescence microscope. (D) Whole-cell lysates were used for Western blotting with antibodies specific for CHOP, BAX, BCL-2, BIM and β-actin (loading control). This experiment was performed in triplicate. (E) The band intensities for CHOP, BCL-2/BAX and BIM were expressed as a histogram relative to β-actin. The control group (no MMC treatment) was normalized to a value of 1.0-fold. The data in panels A, B, C and E are the mean ± SD of at least three independent experiments. *P

    Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.

    Techniques: Transfection, CCK-8 Assay, Double Staining, TUNEL Assay, Staining, Fluorescence, Microscopy, Western Blot

    The role of three UPR sensors in MMC-induced apoptosis in fibroblasts. Fibroblasts were transfected with the PERK, ATF6, IRE1 or non-targeting lentiviral-mediated shRNAs. (A) The expression levels of the targeted transcripts were determined by Western blotting with PERK, ATF6, IRE1 and β-actin antibodies. The presented data represents the results of two independent experiments. (B) Transfected cells were treated with MMC as described in the text and then cell viability was measured after 48 hours. (C) Apoptosis rates were determined via Annexin V/propidium iodide double staining and are shown in the bar graph. (D) After MMC treatment (0.4 mg/ml, 5 minutes) and incubation for 48 hours, equal amounts of the whole cell lysates were analyzed by Western blotting with antibodies specific for CHOP, BIM, cleaved caspase-3, and β-actin (loading control). This experiment was performed in triplicate. The data presented in panels B and C are the mean ± SD of three independent experiments, *P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    doi: 10.1371/journal.pone.0059330

    Figure Lengend Snippet: The role of three UPR sensors in MMC-induced apoptosis in fibroblasts. Fibroblasts were transfected with the PERK, ATF6, IRE1 or non-targeting lentiviral-mediated shRNAs. (A) The expression levels of the targeted transcripts were determined by Western blotting with PERK, ATF6, IRE1 and β-actin antibodies. The presented data represents the results of two independent experiments. (B) Transfected cells were treated with MMC as described in the text and then cell viability was measured after 48 hours. (C) Apoptosis rates were determined via Annexin V/propidium iodide double staining and are shown in the bar graph. (D) After MMC treatment (0.4 mg/ml, 5 minutes) and incubation for 48 hours, equal amounts of the whole cell lysates were analyzed by Western blotting with antibodies specific for CHOP, BIM, cleaved caspase-3, and β-actin (loading control). This experiment was performed in triplicate. The data presented in panels B and C are the mean ± SD of three independent experiments, *P

    Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.

    Techniques: Transfection, Expressing, Western Blot, Double Staining, Incubation

    Phosphatidylinositol 3-kinase/Akt acts downstream of FAK signaling to regulate apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with LY294002. (A and B) The expression of Akt, pAkt, caspase-3 and c-caspase-3 was examined using western blotting. (C and D) Cell apoptosis was examined using (C) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (D) Annexin V/propidium iodide. (E and F) The expression of FAK and pFAK was examined using western blotting. Scale bar, 200 µm. FAK, focal adhesion kinase; pFAK, phosphorylated FAK; c-caspase-3, cleaved caspase-3; TGFβ, transforming growth factor-β.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway

    doi: 10.3892/etm.2015.2745

    Figure Lengend Snippet: Phosphatidylinositol 3-kinase/Akt acts downstream of FAK signaling to regulate apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with LY294002. (A and B) The expression of Akt, pAkt, caspase-3 and c-caspase-3 was examined using western blotting. (C and D) Cell apoptosis was examined using (C) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (D) Annexin V/propidium iodide. (E and F) The expression of FAK and pFAK was examined using western blotting. Scale bar, 200 µm. FAK, focal adhesion kinase; pFAK, phosphorylated FAK; c-caspase-3, cleaved caspase-3; TGFβ, transforming growth factor-β.

    Article Snippet: The treated cells were stained with FITC, Annexin V and propidium iodide (PI), and the stained cells were analyzed using a FACSort™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and evaluated with the CellQuest™ software system (BD Biosciences).

    Techniques: Expressing, Western Blot, End Labeling

    Knockdown of FAK induces apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were transfected with small interfering RNA against FAK (siFAK) or control (conRNA). (A and B) The expression of FAK was examined using western blotting. (C and D) Cell apoptosis was examined using (C) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (D) Annexin V/propidium iodide. (E) Cell survival was examined using an MTT assay. Scale bar, 200 µm. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway

    doi: 10.3892/etm.2015.2745

    Figure Lengend Snippet: Knockdown of FAK induces apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were transfected with small interfering RNA against FAK (siFAK) or control (conRNA). (A and B) The expression of FAK was examined using western blotting. (C and D) Cell apoptosis was examined using (C) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (D) Annexin V/propidium iodide. (E) Cell survival was examined using an MTT assay. Scale bar, 200 µm. *P

    Article Snippet: The treated cells were stained with FITC, Annexin V and propidium iodide (PI), and the stained cells were analyzed using a FACSort™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and evaluated with the CellQuest™ software system (BD Biosciences).

    Techniques: Transfection, Small Interfering RNA, Expressing, Western Blot, End Labeling, MTT Assay

    Src is an important mediator of FAK-regulated apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with PP2. (A and B) Cell apoptosis was examined using (A) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (B) Annexin V/propidium iodide. (C and D) The expression of FAK, pFAK, Src and pSrc was examined using western blotting. Scale bar, 200 µm. FAK, focal adhesion kinase; pFAK, phosphorylated FAK; TGFβ, transforming growth factor-β.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway

    doi: 10.3892/etm.2015.2745

    Figure Lengend Snippet: Src is an important mediator of FAK-regulated apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with PP2. (A and B) Cell apoptosis was examined using (A) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (B) Annexin V/propidium iodide. (C and D) The expression of FAK, pFAK, Src and pSrc was examined using western blotting. Scale bar, 200 µm. FAK, focal adhesion kinase; pFAK, phosphorylated FAK; TGFβ, transforming growth factor-β.

    Article Snippet: The treated cells were stained with FITC, Annexin V and propidium iodide (PI), and the stained cells were analyzed using a FACSort™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and evaluated with the CellQuest™ software system (BD Biosciences).

    Techniques: End Labeling, Expressing, Western Blot

    Suppression of FAK phosphorylation induces apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with PF-228 and 5 ng/ml TGFβ. (A and B) Cell apoptosis was examined using (A) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (B) Annexin V/propidium iodide. (C and D) The expression of FAK, pFAK, Src, pSrc, Akt, pAkt, caspase-3 and c-caspase-3 was examined using western blotting. (E) Cell survival was examined using an MTT assay. Scale bar, 200 µm. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway

    doi: 10.3892/etm.2015.2745

    Figure Lengend Snippet: Suppression of FAK phosphorylation induces apoptosis in T24 bladder cancer cells. T24 bladder cancer cells were treated with PF-228 and 5 ng/ml TGFβ. (A and B) Cell apoptosis was examined using (A) deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and (B) Annexin V/propidium iodide. (C and D) The expression of FAK, pFAK, Src, pSrc, Akt, pAkt, caspase-3 and c-caspase-3 was examined using western blotting. (E) Cell survival was examined using an MTT assay. Scale bar, 200 µm. *P

    Article Snippet: The treated cells were stained with FITC, Annexin V and propidium iodide (PI), and the stained cells were analyzed using a FACSort™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and evaluated with the CellQuest™ software system (BD Biosciences).

    Techniques: End Labeling, Expressing, Western Blot, MTT Assay

    Neutralization of the PM-MSC complex . (a, b) Viability of rat fetal membrane-derived MSCs at 10 min after mixture of PM (final dose 0.8%) with or without neutralization using NaOH in-vitro was assessed by propidium iodide staining. Representative pictures in each group are presented (a). Scale bar = 50 μm. The bar graph presents the averaged data (b). n=4 in each point ; * p

    Journal: Biomaterials

    Article Title: Self-assembling peptide hydrogel enables instant epicardial coating of the heart with mesenchymal stromal cells for the treatment of heart failure

    doi: 10.1016/j.biomaterials.2017.10.050

    Figure Lengend Snippet: Neutralization of the PM-MSC complex . (a, b) Viability of rat fetal membrane-derived MSCs at 10 min after mixture of PM (final dose 0.8%) with or without neutralization using NaOH in-vitro was assessed by propidium iodide staining. Representative pictures in each group are presented (a). Scale bar = 50 μm. The bar graph presents the averaged data (b). n=4 in each point ; * p

    Article Snippet: 5 μl of propidium iodide (PI) solution (Calbiochem, UK) was added to the PM-MSC complex.

    Techniques: Neutralization, Derivative Assay, In Vitro, Staining

    Production and characterization of modified PM . (a) . Appearance and microimage of produced homogenous, neutral PM are shown. Scale bar = 50 μm. (b, c) . Viability of rat fetal membrane-derived MSCs at 10 min after mixture with modified PM (mod-PM; final dose 0.8%) in-vitro was assessed by propidium iodide staining (b). n=4 in each point. Representative pictures of propidium iodide in each group are presented in (c). Scale bar = 50 μm. (d) . Retention of the modified PM-MSC complex was assessed by a tilting test on the plastic plate in-vitro. n=4 in each point. (e) . Retention of the modified PM-MSC complex on the beating rat heart in-vivo. Retained modified PM-MSC complex (white) at 0 min dissociated by 60 min after epicardial placement. Representative pictures from n = 4 are presented.

    Journal: Biomaterials

    Article Title: Self-assembling peptide hydrogel enables instant epicardial coating of the heart with mesenchymal stromal cells for the treatment of heart failure

    doi: 10.1016/j.biomaterials.2017.10.050

    Figure Lengend Snippet: Production and characterization of modified PM . (a) . Appearance and microimage of produced homogenous, neutral PM are shown. Scale bar = 50 μm. (b, c) . Viability of rat fetal membrane-derived MSCs at 10 min after mixture with modified PM (mod-PM; final dose 0.8%) in-vitro was assessed by propidium iodide staining (b). n=4 in each point. Representative pictures of propidium iodide in each group are presented in (c). Scale bar = 50 μm. (d) . Retention of the modified PM-MSC complex was assessed by a tilting test on the plastic plate in-vitro. n=4 in each point. (e) . Retention of the modified PM-MSC complex on the beating rat heart in-vivo. Retained modified PM-MSC complex (white) at 0 min dissociated by 60 min after epicardial placement. Representative pictures from n = 4 are presented.

    Article Snippet: 5 μl of propidium iodide (PI) solution (Calbiochem, UK) was added to the PM-MSC complex.

    Techniques: Modification, Produced, Derivative Assay, In Vitro, Staining, In Vivo

    Cell apoptosis analysis in HEPG2 and MHCC97-H liver cancer cells using Annexin V-FITC/PI staining and flow cytometry. (A) Cell apoptosis analysis by flow cytometry. Early-stage apoptotic cells are presented in the lower right quadrants (Annexin V-FITC positive and PI negative) and late-stage apoptotic cells are presented in the upper right quadrants (Annexin V-FITC and PI positive) (B) Calculated apoptotic rate (%) of early and late stage apoptotic cells. Similar results were obtained in three independent experiments. Results are presented as the mean ± standard error of the mean. FITC, fluorescein isothiocyanate; PI, propidium iodide; siRNA, small interfering RNA.

    Journal: Molecular Medicine Reports

    Article Title: Investigation of the role of cullin 4A overexpression in human liver cancer

    doi: 10.3892/mmr.2018.9233

    Figure Lengend Snippet: Cell apoptosis analysis in HEPG2 and MHCC97-H liver cancer cells using Annexin V-FITC/PI staining and flow cytometry. (A) Cell apoptosis analysis by flow cytometry. Early-stage apoptotic cells are presented in the lower right quadrants (Annexin V-FITC positive and PI negative) and late-stage apoptotic cells are presented in the upper right quadrants (Annexin V-FITC and PI positive) (B) Calculated apoptotic rate (%) of early and late stage apoptotic cells. Similar results were obtained in three independent experiments. Results are presented as the mean ± standard error of the mean. FITC, fluorescein isothiocyanate; PI, propidium iodide; siRNA, small interfering RNA.

    Article Snippet: The cell samples were stained with 200 µl propidium iodide (PI; Beyotime Institute of Biotechnology, Haimen, China) in the presence of RNase A (Beyotime Institute of Biotechnology) for 10 min at room temperature.

    Techniques: Staining, Flow Cytometry, Cytometry, Small Interfering RNA

    Effect of CUL4A on cell proliferation ability. (A) Evaluation of the effects of CUL4A overexpression and CUL4A knockdown on the cell proliferation of HEPG2 and MHCC97-H liver cancer cells using a Cell Counting Kit-8 assay. (B) Propidium iodide staining followed by flow cytometry was performed to analyze the cell cycle distribution in control, CUL4A overexpression and CUL4A-siRNA HEPG2 and MHCC97-H cells. (C) Percentage of HEPG2 or MHCC97-H cells in each phase. Similar results were obtained in three independent experiments. Results are presented as the mean ± standard error of the mean. *P

    Journal: Molecular Medicine Reports

    Article Title: Investigation of the role of cullin 4A overexpression in human liver cancer

    doi: 10.3892/mmr.2018.9233

    Figure Lengend Snippet: Effect of CUL4A on cell proliferation ability. (A) Evaluation of the effects of CUL4A overexpression and CUL4A knockdown on the cell proliferation of HEPG2 and MHCC97-H liver cancer cells using a Cell Counting Kit-8 assay. (B) Propidium iodide staining followed by flow cytometry was performed to analyze the cell cycle distribution in control, CUL4A overexpression and CUL4A-siRNA HEPG2 and MHCC97-H cells. (C) Percentage of HEPG2 or MHCC97-H cells in each phase. Similar results were obtained in three independent experiments. Results are presented as the mean ± standard error of the mean. *P

    Article Snippet: The cell samples were stained with 200 µl propidium iodide (PI; Beyotime Institute of Biotechnology, Haimen, China) in the presence of RNase A (Beyotime Institute of Biotechnology) for 10 min at room temperature.

    Techniques: Over Expression, Cell Counting, Staining, Flow Cytometry, Cytometry

    PCB-induced arrest is reversible and cells regain sensitivity to VCR after reentering the cell cycle. A . ALL-5 and T98G cells were stained with propidium iodide and subjected to two-parameter flow cytometry to create a gate distinguishing EdU-negative and EdU-positive cells (left panels). ALL-5 and T98G cells were then treated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), washed thrice with PBS, and incubated in growth media containing 10 µM EdU for the times indicated. Cells were harvested, fixed, and stained for EdU incorporation and with propidium iodide and analyzed by two-parameter flow cytometry. Data shown are representative of three independent experiments. The percentage of cells positive for EdU is indicated at the top of each panel. B . ALL-5 and T98G cells were treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h with or without pretreatment with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G). A third set of cells were pretreated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), after which cells were washed thrice with PBS and treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h. Cells were stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: PCB-induced arrest is reversible and cells regain sensitivity to VCR after reentering the cell cycle. A . ALL-5 and T98G cells were stained with propidium iodide and subjected to two-parameter flow cytometry to create a gate distinguishing EdU-negative and EdU-positive cells (left panels). ALL-5 and T98G cells were then treated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), washed thrice with PBS, and incubated in growth media containing 10 µM EdU for the times indicated. Cells were harvested, fixed, and stained for EdU incorporation and with propidium iodide and analyzed by two-parameter flow cytometry. Data shown are representative of three independent experiments. The percentage of cells positive for EdU is indicated at the top of each panel. B . ALL-5 and T98G cells were treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h with or without pretreatment with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G). A third set of cells were pretreated with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G), after which cells were washed thrice with PBS and treated with 0.1% DMSO or 100 nM (ALL-5) or 1 µM (T98G) VCR for 48 h. Cells were stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Incubation

    Vinblastine induces death in G1 phase which is prevented by PCB pretreatment. A . ALL-5 cells in G1 phase were isolated using centrifugal elutriation and treated with 0.1% DMSO or 100 nM vinblastine (VBL) for the times indicated. Cells were fixed and stained with propidium iodide and DNA content analyzed by flow cytometry. Data shown are representative of two independent experiments of three replicates each. B . ALL-5 cells were treated with 0.1% DMSO or 100 nM VBL for 48 h with or without pretreatment with 1 µM PCB for 72 h. Cells were fixed and stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: Vinblastine induces death in G1 phase which is prevented by PCB pretreatment. A . ALL-5 cells in G1 phase were isolated using centrifugal elutriation and treated with 0.1% DMSO or 100 nM vinblastine (VBL) for the times indicated. Cells were fixed and stained with propidium iodide and DNA content analyzed by flow cytometry. Data shown are representative of two independent experiments of three replicates each. B . ALL-5 cells were treated with 0.1% DMSO or 100 nM VBL for 48 h with or without pretreatment with 1 µM PCB for 72 h. Cells were fixed and stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Isolation, Staining, Flow Cytometry, Cytometry

    Cycloheximide treated ALL cells arrest in G1 phase and become insensitive to VCR. A,B . ALL-5 cells isolated in G1 phase by centrifugal elutriation were treated with 1 µg/mL cycloheximide (CHX) or 100 nM VCR or both and stained with propidium iodide and analyzed for DNA content by flow cytometry. The percentage of cells with 2N DNA (G1 phase) ( A ) or sub-G1 DNA ( B ) are shown (mean ± S.D., n = 3). C . Whole cell extracts were prepared from G1 phase ALL-5 cells treated with 1 µM PCB or 1 µg/mL CHX for the times indicated and subjected to immunoblot analysis for RB, phospho-RB (Ser 807/811), cyclin D, or GAPDH as a loading control.

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: Cycloheximide treated ALL cells arrest in G1 phase and become insensitive to VCR. A,B . ALL-5 cells isolated in G1 phase by centrifugal elutriation were treated with 1 µg/mL cycloheximide (CHX) or 100 nM VCR or both and stained with propidium iodide and analyzed for DNA content by flow cytometry. The percentage of cells with 2N DNA (G1 phase) ( A ) or sub-G1 DNA ( B ) are shown (mean ± S.D., n = 3). C . Whole cell extracts were prepared from G1 phase ALL-5 cells treated with 1 µM PCB or 1 µg/mL CHX for the times indicated and subjected to immunoblot analysis for RB, phospho-RB (Ser 807/811), cyclin D, or GAPDH as a loading control.

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Isolation, Staining, Flow Cytometry, Cytometry

    PCB causes G1 phase arrest in ALL-5 and T98G but not HeLa cells. A . Cells were treated with vehicle (0.1% DMSO) or 1 µM PCB for 72 h (ALL-5) or 48 h (T98G and HeLa) and DNA content determined by propidium iodide staining and flow cytometry. Data shown are mean ± S.D. (n = 4). B . ALL-5 or T98G cells were treated with 1 μM PCB for 72 h or 48 h, respectively. Cells were fixed and stained for Ki-67 (red) or with DAPI (blue) as a nuclear marker. The scale bar in ALL-5 images is 60 μm while that in T98G images is 120 μm. Images are representative of six fields of vision. C . Mean intensity fluorescence of Ki-67 was quantified for 20 cells per six fields of vision using ImageJ. Cells were selected using DAPI stain while blind to Ki-67 intensity. Each point represents a single cell while horizontal bars represent the mean ± S.D. of all data points (n = 120).

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: PCB causes G1 phase arrest in ALL-5 and T98G but not HeLa cells. A . Cells were treated with vehicle (0.1% DMSO) or 1 µM PCB for 72 h (ALL-5) or 48 h (T98G and HeLa) and DNA content determined by propidium iodide staining and flow cytometry. Data shown are mean ± S.D. (n = 4). B . ALL-5 or T98G cells were treated with 1 μM PCB for 72 h or 48 h, respectively. Cells were fixed and stained for Ki-67 (red) or with DAPI (blue) as a nuclear marker. The scale bar in ALL-5 images is 60 μm while that in T98G images is 120 μm. Images are representative of six fields of vision. C . Mean intensity fluorescence of Ki-67 was quantified for 20 cells per six fields of vision using ImageJ. Cells were selected using DAPI stain while blind to Ki-67 intensity. Each point represents a single cell while horizontal bars represent the mean ± S.D. of all data points (n = 120).

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Marker, Fluorescence

    ALL-5 and T98G cells are refractory to VCR after PCB pretreatment while HeLa cells retain their sensitivity. A . ALL-5, T98G, or HeLa cells were treated with 0.1% DMSO or 100 nM (ALL-5 and HeLa) or 1 µM (T98G cells) VCR for 48 h with or without pretreatment with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G and HeLa). Cells were subjected to propidium iodide staining and flow cytometry to determine the percentage of cells with

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: ALL-5 and T98G cells are refractory to VCR after PCB pretreatment while HeLa cells retain their sensitivity. A . ALL-5, T98G, or HeLa cells were treated with 0.1% DMSO or 100 nM (ALL-5 and HeLa) or 1 µM (T98G cells) VCR for 48 h with or without pretreatment with 1 µM PCB for 72 h (ALL-5) or 48 h (T98G and HeLa). Cells were subjected to propidium iodide staining and flow cytometry to determine the percentage of cells with

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Staining, Flow Cytometry, Cytometry

    Pretreatment with PCB does not prevent VCR from disrupting the microtubule network or block downstream apoptotic signaling. A . HeLa or ALL-5 cells were treated with 0.1% DMSO or 100 nM vincristine (VCR) for 14 h, with or without pretreatment with 1 µM PCB for 72 h, as indicated. Cells were fixed and permeabilized and α-tubulin visualized by fluorescence microscopy. B . ALL-5 cells were treated with 0.1% DMSO or 100 nM ABT-263 for 16 h with or without pretreatment with 1 µM PCB for 72 h. Cells were fixed and stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Journal: Cell Cycle

    Article Title: Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

    doi: 10.1080/15384101.2018.1496746

    Figure Lengend Snippet: Pretreatment with PCB does not prevent VCR from disrupting the microtubule network or block downstream apoptotic signaling. A . HeLa or ALL-5 cells were treated with 0.1% DMSO or 100 nM vincristine (VCR) for 14 h, with or without pretreatment with 1 µM PCB for 72 h, as indicated. Cells were fixed and permeabilized and α-tubulin visualized by fluorescence microscopy. B . ALL-5 cells were treated with 0.1% DMSO or 100 nM ABT-263 for 16 h with or without pretreatment with 1 µM PCB for 72 h. Cells were fixed and stained with propidium iodide and subjected to flow cytometry to determine the percentage of cells with

    Article Snippet: DNA content was evaluated by propidium iodide staining according to the manufacturer’s instructions (BD Pharmigen) and flow cytometry was carried out by the UAMS Flow Cytometry Core Facility using a FACSCalibur (Becton Dickinson, Mountain View, CA).

    Techniques: Blocking Assay, Fluorescence, Microscopy, Staining, Flow Cytometry, Cytometry

    Glucose inhibition of differentiation is independent of glycolysis. (A) Outgrowth and (B) cellular viability of SS cultures after treatment with 5 mM indicated compounds (xyl, xylose; 2-DOG, 2-deoxyglucose; 6-DOG, 6-deoxyglucose). SS parasites were washed and resuspended (5 × 10 5 /ml) in RPMIθ with proline and threonine for the first 2 days and then transferred to the very-low-glucose PF medium SDM79θ supplemented with 5 mM indicated compound, and growth was monitored. (The transfer was necessary to provide amino acids required for PF viability.) (C) Impact of varied levels of 6-DOG on SS outgrowth and (D) viability by flow cytometry and propidium iodide (PI) staining. Concentrations of 6-DOG were maintained throughout the experiment. Bars indicate standard deviation in triplicate assays.

    Journal: mSphere

    Article Title: Glucose Signaling Is Important for Nutrient Adaptation during Differentiation of Pleomorphic African Trypanosomes

    doi: 10.1128/mSphere.00366-18

    Figure Lengend Snippet: Glucose inhibition of differentiation is independent of glycolysis. (A) Outgrowth and (B) cellular viability of SS cultures after treatment with 5 mM indicated compounds (xyl, xylose; 2-DOG, 2-deoxyglucose; 6-DOG, 6-deoxyglucose). SS parasites were washed and resuspended (5 × 10 5 /ml) in RPMIθ with proline and threonine for the first 2 days and then transferred to the very-low-glucose PF medium SDM79θ supplemented with 5 mM indicated compound, and growth was monitored. (The transfer was necessary to provide amino acids required for PF viability.) (C) Impact of varied levels of 6-DOG on SS outgrowth and (D) viability by flow cytometry and propidium iodide (PI) staining. Concentrations of 6-DOG were maintained throughout the experiment. Bars indicate standard deviation in triplicate assays.

    Article Snippet: Cell numbers were scored daily during the first week and every other-day in the second week, and cell viability was determined by propidium iodide (PI) staining (0.5 μg/ml final concentration) followed by flow cytometry on an Accuri BD flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Inhibition, Flow Cytometry, Cytometry, Staining, Standard Deviation

    Slender form (LS) and short stumpy form (SS) T. brucei rapidly deplete glucose. Parasites isolated from rodent buffy coats by chromatography were washed extensively in PBS and then resuspended (4 × 10 5 cells/ml for LS and 5 × 10 5 cells/ml for SS) in RPMIθ supplemented with proline and threonine and different concentrations of glucose. LS parasites (left column) were isolated after 4 days of infection and made up nearly 100% of the parasite population as determined by microscopy, while the SS samples (right column) contained a mixture of SS (∼90%) and LS (∼10%) parasites as scored by cytometry of PAD1-labeled parasites (not shown). (A) Glucose concentrations in the medium were measured through time as described in Materials and Methods and standard deviations from experiments performed in triplicate are indicated. (B) Parasite viability was scored by propidium iodide staining.

    Journal: mSphere

    Article Title: Glucose Signaling Is Important for Nutrient Adaptation during Differentiation of Pleomorphic African Trypanosomes

    doi: 10.1128/mSphere.00366-18

    Figure Lengend Snippet: Slender form (LS) and short stumpy form (SS) T. brucei rapidly deplete glucose. Parasites isolated from rodent buffy coats by chromatography were washed extensively in PBS and then resuspended (4 × 10 5 cells/ml for LS and 5 × 10 5 cells/ml for SS) in RPMIθ supplemented with proline and threonine and different concentrations of glucose. LS parasites (left column) were isolated after 4 days of infection and made up nearly 100% of the parasite population as determined by microscopy, while the SS samples (right column) contained a mixture of SS (∼90%) and LS (∼10%) parasites as scored by cytometry of PAD1-labeled parasites (not shown). (A) Glucose concentrations in the medium were measured through time as described in Materials and Methods and standard deviations from experiments performed in triplicate are indicated. (B) Parasite viability was scored by propidium iodide staining.

    Article Snippet: Cell numbers were scored daily during the first week and every other-day in the second week, and cell viability was determined by propidium iodide (PI) staining (0.5 μg/ml final concentration) followed by flow cytometry on an Accuri BD flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Isolation, Chromatography, Infection, Microscopy, Cytometry, Labeling, Staining

    Amino acids are required for completion of SS differentiation and cell viability in very-low-glucose medium. SS parasites cultured in bloodstream form very-low-glucose medium, RPMIθ (which allowed manipulation of amino acid content) supplemented with or without glucose, proline (P, 4.6 mM), threonine (T, 3.4 mM), or proline and threonine for 2 days followed by transfer to amino acid-replete SDM79θ were scored for (A) outgrowth and (B) viability by flow cytometry and propidium iodide (PI) staining. (C) Assessment of parasite outgrowth and (D) viability after oxidative phosphorylation inhibition by treatment with oligomycin (either 100 ng/ml or 1 μg/ml) for 2 days in RPMIθ with proline and threonine (AA) followed by transfer to SDM79θ to provide amino acids for PF parasites. Bars indicate standard deviation in triplicate assays.

    Journal: mSphere

    Article Title: Glucose Signaling Is Important for Nutrient Adaptation during Differentiation of Pleomorphic African Trypanosomes

    doi: 10.1128/mSphere.00366-18

    Figure Lengend Snippet: Amino acids are required for completion of SS differentiation and cell viability in very-low-glucose medium. SS parasites cultured in bloodstream form very-low-glucose medium, RPMIθ (which allowed manipulation of amino acid content) supplemented with or without glucose, proline (P, 4.6 mM), threonine (T, 3.4 mM), or proline and threonine for 2 days followed by transfer to amino acid-replete SDM79θ were scored for (A) outgrowth and (B) viability by flow cytometry and propidium iodide (PI) staining. (C) Assessment of parasite outgrowth and (D) viability after oxidative phosphorylation inhibition by treatment with oligomycin (either 100 ng/ml or 1 μg/ml) for 2 days in RPMIθ with proline and threonine (AA) followed by transfer to SDM79θ to provide amino acids for PF parasites. Bars indicate standard deviation in triplicate assays.

    Article Snippet: Cell numbers were scored daily during the first week and every other-day in the second week, and cell viability was determined by propidium iodide (PI) staining (0.5 μg/ml final concentration) followed by flow cytometry on an Accuri BD flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Staining, Inhibition, Standard Deviation

    Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by propidium iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P

    Journal: Scientific Reports

    Article Title: Inhibitory effects of isocryptotanshinone on gastric cancer

    doi: 10.1038/s41598-018-27638-0

    Figure Lengend Snippet: Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by propidium iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P

    Article Snippet: Cell Counting kit (CCK)−8 and propidium iodide (PI) were obtained from Beyotime, and FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences.

    Techniques: Concentration Assay, Staining, Flow Cytometry, Cytometry, Software

    Effect of isocryptotanshinone on apoptosis of SGC-7901 and MKN-45 cells. Cells were exposed to the indicated concentration of ICTS for 24 hours, and the SGC-7901 ( A ) and MKN-45 ( D ) cells stained using propidium iodide and annexin-v were detected using FACScan flow cytometry. The percentages of apoptotic SGC-7901 ( B ) and MKN-45 ( E ) cells were determined by the Flow Jo software and outputted in histogram. Expression of cleaved PARP and cleaved caspase-9 in SGC-7901 ( C ) and MKN-45 ( F ) cells were determined by western blot. Data were expressed as mean ± standard error of triplicates of one representative experiment. β-actin was used as the loading control. ICTS, isocryptotanshinone.

    Journal: Scientific Reports

    Article Title: Inhibitory effects of isocryptotanshinone on gastric cancer

    doi: 10.1038/s41598-018-27638-0

    Figure Lengend Snippet: Effect of isocryptotanshinone on apoptosis of SGC-7901 and MKN-45 cells. Cells were exposed to the indicated concentration of ICTS for 24 hours, and the SGC-7901 ( A ) and MKN-45 ( D ) cells stained using propidium iodide and annexin-v were detected using FACScan flow cytometry. The percentages of apoptotic SGC-7901 ( B ) and MKN-45 ( E ) cells were determined by the Flow Jo software and outputted in histogram. Expression of cleaved PARP and cleaved caspase-9 in SGC-7901 ( C ) and MKN-45 ( F ) cells were determined by western blot. Data were expressed as mean ± standard error of triplicates of one representative experiment. β-actin was used as the loading control. ICTS, isocryptotanshinone.

    Article Snippet: Cell Counting kit (CCK)−8 and propidium iodide (PI) were obtained from Beyotime, and FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences.

    Techniques: Concentration Assay, Staining, Flow Cytometry, Cytometry, Software, Expressing, Western Blot

    Baricitinib suppresses CD80/CD86 expression on dendritic cells (DCs). Immature monocyte-derived dendritic cells were cultured with or without baricitinib and tofacitinib during lipopolysaccharide stimulation for 48 h. The DC phenotype was evaluated using flow cytometry. (A) Expression of HLA-DR, CD80, and CD86. (B) Representative histogram data of HLA-DR, CD80, and CD86 expression. (C) Rate of viable cells (annexin V neg /propidium iodide neg ). (D) Expression of CD80 and CD86 in the presence of baricitinib and tofacitinib. Data are mean ± SD of three different donors per group. * p

    Journal: Frontiers in Immunology

    Article Title: Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System

    doi: 10.3389/fimmu.2018.01510

    Figure Lengend Snippet: Baricitinib suppresses CD80/CD86 expression on dendritic cells (DCs). Immature monocyte-derived dendritic cells were cultured with or without baricitinib and tofacitinib during lipopolysaccharide stimulation for 48 h. The DC phenotype was evaluated using flow cytometry. (A) Expression of HLA-DR, CD80, and CD86. (B) Representative histogram data of HLA-DR, CD80, and CD86 expression. (C) Rate of viable cells (annexin V neg /propidium iodide neg ). (D) Expression of CD80 and CD86 in the presence of baricitinib and tofacitinib. Data are mean ± SD of three different donors per group. * p

    Article Snippet: Cell viability was evaluated by Annexin V and Propidium Iodide (BioLegend, San Diego, CA, USA).

    Techniques: Expressing, Derivative Assay, Cell Culture, Flow Cytometry, Cytometry

    Baricitinib suppresses type-I interferon (IFN) production from plasmacytoid dendritic cells (pDCs). Peripheral blood mononuclear cells were stimulated with toll-like receptor (TLR) 9 agonist with or without baricitinib and other inhibitors. TLR9-mediated cytokine production by pDCs (Lin − HLA-DR + CD11c − CD123 + cells) was measured by intracellular staining or ELISA. (A) Percentage of TNF-α- and IFN-α-producing pDCs. (B) Representative flow cytometry plots showing IFN-α production by pDCs. (C) IFN-α concentration in the supernatants. (D) Percentage of TNF-α- and IFN-α-producing pDCs in the presence of baricitinib and other inhibitors. (E) Representative histogram data of annexin V/propidium iodide staining from three independent experiments. (F) Expression of CD80 and CD86 on pDCs. Data are mean ± SD of three different donors per group. * p

    Journal: Frontiers in Immunology

    Article Title: Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System

    doi: 10.3389/fimmu.2018.01510

    Figure Lengend Snippet: Baricitinib suppresses type-I interferon (IFN) production from plasmacytoid dendritic cells (pDCs). Peripheral blood mononuclear cells were stimulated with toll-like receptor (TLR) 9 agonist with or without baricitinib and other inhibitors. TLR9-mediated cytokine production by pDCs (Lin − HLA-DR + CD11c − CD123 + cells) was measured by intracellular staining or ELISA. (A) Percentage of TNF-α- and IFN-α-producing pDCs. (B) Representative flow cytometry plots showing IFN-α production by pDCs. (C) IFN-α concentration in the supernatants. (D) Percentage of TNF-α- and IFN-α-producing pDCs in the presence of baricitinib and other inhibitors. (E) Representative histogram data of annexin V/propidium iodide staining from three independent experiments. (F) Expression of CD80 and CD86 on pDCs. Data are mean ± SD of three different donors per group. * p

    Article Snippet: Cell viability was evaluated by Annexin V and Propidium Iodide (BioLegend, San Diego, CA, USA).

    Techniques: Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Concentration Assay, Expressing

    Baricitinib inhibits B cell differentiation. Human B cells were purified and cultured for 5 days in the presence of anti-human IgM Ab F(ab′)2 fragments and under interferon (IFN)-α stimulation. Plasmablast (CD3 − CD19 + CD20 − CD27 hi CD38 hi ) differentiation was assessed by flow cytometry, and IgG Ab titers and interleukin (IL)-6 concentrations in culture supernatants were measured by ELISA Cytometric Bead Array. (A) Percentage of plasmablasts. (B) Representative flow cytometry plots showing plasmablasts. (C) Percentage of plasmablast, IL-6, and IgG concentrations in the presence of baricitinib and other inhibitors. (D) Representative histogram data of annexin V/propidium iodide staining. (E) Rate of viable cells (annexin V neg /propidium iodide neg ). Data are mean ± SD of three different donors per group. * p

    Journal: Frontiers in Immunology

    Article Title: Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System

    doi: 10.3389/fimmu.2018.01510

    Figure Lengend Snippet: Baricitinib inhibits B cell differentiation. Human B cells were purified and cultured for 5 days in the presence of anti-human IgM Ab F(ab′)2 fragments and under interferon (IFN)-α stimulation. Plasmablast (CD3 − CD19 + CD20 − CD27 hi CD38 hi ) differentiation was assessed by flow cytometry, and IgG Ab titers and interleukin (IL)-6 concentrations in culture supernatants were measured by ELISA Cytometric Bead Array. (A) Percentage of plasmablasts. (B) Representative flow cytometry plots showing plasmablasts. (C) Percentage of plasmablast, IL-6, and IgG concentrations in the presence of baricitinib and other inhibitors. (D) Representative histogram data of annexin V/propidium iodide staining. (E) Rate of viable cells (annexin V neg /propidium iodide neg ). Data are mean ± SD of three different donors per group. * p

    Article Snippet: Cell viability was evaluated by Annexin V and Propidium Iodide (BioLegend, San Diego, CA, USA).

    Techniques: Cell Differentiation, Purification, Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Staining

    Radioresistant TE-1 and Eca-109 cells showed more aggressive malignancies. (A) Wound-healing assay of TE-1 and TE-1/R cells. (B) Wound-healing assay of Eca-109 and Eca-109/R cells. Wound healing was observed 24 h after the treatment. (C) Induction of apoptosis by radiation in TE-1 and TE-1/R cells. (D) Induction of apoptosis by radiation in Eca-109 and Eca-109/R cells. Cells were treated with 6 Gy irradiation, and the apoptosis was measured using propidium iodide (PI)/Annexin-V double staining. Data are normalized to the control cells and presented as the mean ± SEM of three independent experiments, * P

    Journal: Journal of Cancer

    Article Title: mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

    doi: 10.7150/jca.15652

    Figure Lengend Snippet: Radioresistant TE-1 and Eca-109 cells showed more aggressive malignancies. (A) Wound-healing assay of TE-1 and TE-1/R cells. (B) Wound-healing assay of Eca-109 and Eca-109/R cells. Wound healing was observed 24 h after the treatment. (C) Induction of apoptosis by radiation in TE-1 and TE-1/R cells. (D) Induction of apoptosis by radiation in Eca-109 and Eca-109/R cells. Cells were treated with 6 Gy irradiation, and the apoptosis was measured using propidium iodide (PI)/Annexin-V double staining. Data are normalized to the control cells and presented as the mean ± SEM of three independent experiments, * P

    Article Snippet: Apoptosis was measured using propidium iodide (PI)/Annexin-V double staining following the manufacturer's instructions (Keygen Biotech, Nanjing, China).

    Techniques: Wound Healing Assay, Irradiation, Double Staining

    The effect of Sall2 overexpression on the apoptosis, migration and cisplatin sensitivity of esophageal cancer cells. (A) Cells were transiently transfected with control vector or Sall2-overexpression vector (pcDNA3.1-Sall2). The expression of Sall2 was validated by Western blot. Apoptosis of (B) TE-1/R and (C) Eca-109/R cells was measured using propidium iodide (PI)/Annexin-V double staining. (D) Wound-healing assay of cells after the indicated transfection in TE-1/R cells. (E) Wound-healing assay of Eca-109/R cells after the indicated transfection. Wound-healing was observed 24 h after the treatment. (F) TE-1 and (G) Eca-109 cells were treated with cisplatin for 24 h. MTT assay of cell viability. The data are shown as the mean ± SEM for three independent experiments. Statistical analysis between the groups was determined by ANOVA; * P

    Journal: Journal of Cancer

    Article Title: mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

    doi: 10.7150/jca.15652

    Figure Lengend Snippet: The effect of Sall2 overexpression on the apoptosis, migration and cisplatin sensitivity of esophageal cancer cells. (A) Cells were transiently transfected with control vector or Sall2-overexpression vector (pcDNA3.1-Sall2). The expression of Sall2 was validated by Western blot. Apoptosis of (B) TE-1/R and (C) Eca-109/R cells was measured using propidium iodide (PI)/Annexin-V double staining. (D) Wound-healing assay of cells after the indicated transfection in TE-1/R cells. (E) Wound-healing assay of Eca-109/R cells after the indicated transfection. Wound-healing was observed 24 h after the treatment. (F) TE-1 and (G) Eca-109 cells were treated with cisplatin for 24 h. MTT assay of cell viability. The data are shown as the mean ± SEM for three independent experiments. Statistical analysis between the groups was determined by ANOVA; * P

    Article Snippet: Apoptosis was measured using propidium iodide (PI)/Annexin-V double staining following the manufacturer's instructions (Keygen Biotech, Nanjing, China).

    Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Expressing, Western Blot, Double Staining, Wound Healing Assay, MTT Assay

    Confocal microscopy of MG-63 cells seeded on ST1 and ST2 - day 14. Confocal microscopy of MG-63 cells seeded on ST1 ( a , c ) or ST2 ( b , d ) scaffolds from polylactic acid after a 14-day culture. Cells were fixed and cell membranes were stained using DiOC6 (3) ( green ), cell nuclei were stained with propidium iodide ( red ). Both maximum projections ( a - b ) and color coded projections ( c , d ), which display depth ( d ) distribution of cells (d = 180 μm in C, d = 200 μm in D) showed confluent layer of MG-63 cells and formation of bridges from cells connecting fibres on both scaffolds. Objective ×10, magnification ×2, Bar = 50 μm

    Journal: Journal of Biological Engineering

    Article Title: Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

    doi: 10.1186/s13036-017-0074-3

    Figure Lengend Snippet: Confocal microscopy of MG-63 cells seeded on ST1 and ST2 - day 14. Confocal microscopy of MG-63 cells seeded on ST1 ( a , c ) or ST2 ( b , d ) scaffolds from polylactic acid after a 14-day culture. Cells were fixed and cell membranes were stained using DiOC6 (3) ( green ), cell nuclei were stained with propidium iodide ( red ). Both maximum projections ( a - b ) and color coded projections ( c , d ), which display depth ( d ) distribution of cells (d = 180 μm in C, d = 200 μm in D) showed confluent layer of MG-63 cells and formation of bridges from cells connecting fibres on both scaffolds. Objective ×10, magnification ×2, Bar = 50 μm

    Article Snippet: It was then rinsed with PBS (pH 7.4); propidium iodide (5 μg/ml in PBS pH 7.4) was added for 6 min, rinsed with PBS (pH 7.4) The cells were visualized under a confocal microscope (Zeiss LSM 5 DUO) at λexc = 488 nm, λem = 505–550 nm for BCECF-AM and λexc = 560 nm, λem > 570 nm for propidium iodide.

    Techniques: Confocal Microscopy, Staining

    Confocal microscopy of MG-63 cells seeded on ST1 and ST2 - day 3 and day 7. Confocal microscopy of MG-63 cells seeded on ST1 ( a , c , e ) or ST2 ( b , d , f ) scaffolds from polylactic acid after a 3-day culture ( a , b ) or a 7-day culture ( c - f ). Cells were fixed and cell membranes were stained using DiOC6 (3) ( green ), cell nuclei were stained with propidium iodide ( red ). Both maximum projections ( a - d ) and color coded projections ( e , f ), which display depth ( d ) distribution of cells (d = 100 μm in E, d = 400 μm in F) showed fast growth of MG-63 cells on both scaffolds and formation of bridges from cells connecting fibres on ST1 scaffolds on day 7. Objective ×10, Magn. ×2, Bar = 100 μm

    Journal: Journal of Biological Engineering

    Article Title: Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

    doi: 10.1186/s13036-017-0074-3

    Figure Lengend Snippet: Confocal microscopy of MG-63 cells seeded on ST1 and ST2 - day 3 and day 7. Confocal microscopy of MG-63 cells seeded on ST1 ( a , c , e ) or ST2 ( b , d , f ) scaffolds from polylactic acid after a 3-day culture ( a , b ) or a 7-day culture ( c - f ). Cells were fixed and cell membranes were stained using DiOC6 (3) ( green ), cell nuclei were stained with propidium iodide ( red ). Both maximum projections ( a - d ) and color coded projections ( e , f ), which display depth ( d ) distribution of cells (d = 100 μm in E, d = 400 μm in F) showed fast growth of MG-63 cells on both scaffolds and formation of bridges from cells connecting fibres on ST1 scaffolds on day 7. Objective ×10, Magn. ×2, Bar = 100 μm

    Article Snippet: It was then rinsed with PBS (pH 7.4); propidium iodide (5 μg/ml in PBS pH 7.4) was added for 6 min, rinsed with PBS (pH 7.4) The cells were visualized under a confocal microscope (Zeiss LSM 5 DUO) at λexc = 488 nm, λem = 505–550 nm for BCECF-AM and λexc = 560 nm, λem > 570 nm for propidium iodide.

    Techniques: Confocal Microscopy, Staining

    Confocal microscopy photomicrographs of ST1 and ST2 seeded with osteosarcoma cells. Confocal microscopy photomicrographs of ST1 ( a , c ) and ST2 ( b , d ) scaffolds from polylactic acid seeded with osteosarcoma cells MG-63 after a 7-day and 14-day culture. Immunohistochemical staining using monoclonal antibody against either type I collagen ( a , b ) or osteocalcin ( c , d ), followed by secondary antibody conjugated with Alexa Fluor 488® ( green ) and propidium iodide staining of cell nuclei ( red ) showed groups of cells producing type I collagen on both scaffolds ( a , b ) after 7 days, but only rare osteocalcin staining in both scaffolds ( c , d ) after 14 days. Objective ×10×, magnification ×4, bar = 20 μm

    Journal: Journal of Biological Engineering

    Article Title: Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

    doi: 10.1186/s13036-017-0074-3

    Figure Lengend Snippet: Confocal microscopy photomicrographs of ST1 and ST2 seeded with osteosarcoma cells. Confocal microscopy photomicrographs of ST1 ( a , c ) and ST2 ( b , d ) scaffolds from polylactic acid seeded with osteosarcoma cells MG-63 after a 7-day and 14-day culture. Immunohistochemical staining using monoclonal antibody against either type I collagen ( a , b ) or osteocalcin ( c , d ), followed by secondary antibody conjugated with Alexa Fluor 488® ( green ) and propidium iodide staining of cell nuclei ( red ) showed groups of cells producing type I collagen on both scaffolds ( a , b ) after 7 days, but only rare osteocalcin staining in both scaffolds ( c , d ) after 14 days. Objective ×10×, magnification ×4, bar = 20 μm

    Article Snippet: It was then rinsed with PBS (pH 7.4); propidium iodide (5 μg/ml in PBS pH 7.4) was added for 6 min, rinsed with PBS (pH 7.4) The cells were visualized under a confocal microscope (Zeiss LSM 5 DUO) at λexc = 488 nm, λem = 505–550 nm for BCECF-AM and λexc = 560 nm, λem > 570 nm for propidium iodide.

    Techniques: Confocal Microscopy, Immunohistochemistry, Staining

    Effect of combined RQC or individual quercetin on breast cancer cell proliferation. MDA-MB-231 and MDA-MB-435 cells in 5% serum and phenol red-free media were treated with vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each or 15μM quercetin for 48h. Cells were fixed, nuclei stained with Propidium Iodide (PI), and intact (non-apoptotic) nuclei quantified. Percentage of viable cells ± SEM for 30 microscopic fields/triplicate treatments (N = 3) is presented. A) Average cell viability of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. (B) Average cell viability of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.

    Journal: PLoS ONE

    Article Title: Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    doi: 10.1371/journal.pone.0157251

    Figure Lengend Snippet: Effect of combined RQC or individual quercetin on breast cancer cell proliferation. MDA-MB-231 and MDA-MB-435 cells in 5% serum and phenol red-free media were treated with vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each or 15μM quercetin for 48h. Cells were fixed, nuclei stained with Propidium Iodide (PI), and intact (non-apoptotic) nuclei quantified. Percentage of viable cells ± SEM for 30 microscopic fields/triplicate treatments (N = 3) is presented. A) Average cell viability of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. (B) Average cell viability of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.

    Article Snippet: After 8h incubation, cells that migrated through the 8μM pore membrane were fixed with methanol, nuclei stained with Propidium Iodide, and the nuclei were counted from digital images acquired using a microscope (model CKX41; Olympus America, Inc., Center Valley, P.A.)

    Techniques: Multiple Displacement Amplification, Staining

    Effect of combined RQC or individual quercetin on breast cancer cell apoptosis. Quiescent MDA-MB-231 ( A ) or MDA-MB-435 ( B ) cells in 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each, or Quercetin 15μM for 48h. Trypsinized cells were incubated with Annexin V conjugate and propidium iodide, and analyzed by flow cytometry, using a four-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Representative dot plots of 20,000 events/ treatment (N = 3), collected using Cell Quest software 3.3 (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software vX.0.7 (BD Biosciences, San Jose, CA). Cell size and granularity were determined on forward versus side scatter (FSC vs. SSC). Annexin-V fluorescence was measured at 530/30 nm and Propidium Iodide at 585/42nm. The average percentage of early and late apoptotic cells obtained from Annexin V vs. Propidium Iodide dot plots are shown below. (C) Effect of combined RQC or individual quercetin on caspase 3/7 activity. Quiescent MDA-MB-231 cells in 96 well plate at 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each or quercetin 15μM for 48hr or 96hr. Then Caspase-Glo®3/7 reagent were added to each treatment, and after 1hr of incubation the luminescence was measured in a plate-reader luminometer. Relative luminescence to vehicle is shown, N = 3±SEM, asterisk indicates statistical significance (p≤0.05) when compared to vehicle.

    Journal: PLoS ONE

    Article Title: Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    doi: 10.1371/journal.pone.0157251

    Figure Lengend Snippet: Effect of combined RQC or individual quercetin on breast cancer cell apoptosis. Quiescent MDA-MB-231 ( A ) or MDA-MB-435 ( B ) cells in 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each, or Quercetin 15μM for 48h. Trypsinized cells were incubated with Annexin V conjugate and propidium iodide, and analyzed by flow cytometry, using a four-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Representative dot plots of 20,000 events/ treatment (N = 3), collected using Cell Quest software 3.3 (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software vX.0.7 (BD Biosciences, San Jose, CA). Cell size and granularity were determined on forward versus side scatter (FSC vs. SSC). Annexin-V fluorescence was measured at 530/30 nm and Propidium Iodide at 585/42nm. The average percentage of early and late apoptotic cells obtained from Annexin V vs. Propidium Iodide dot plots are shown below. (C) Effect of combined RQC or individual quercetin on caspase 3/7 activity. Quiescent MDA-MB-231 cells in 96 well plate at 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each or quercetin 15μM for 48hr or 96hr. Then Caspase-Glo®3/7 reagent were added to each treatment, and after 1hr of incubation the luminescence was measured in a plate-reader luminometer. Relative luminescence to vehicle is shown, N = 3±SEM, asterisk indicates statistical significance (p≤0.05) when compared to vehicle.

    Article Snippet: After 8h incubation, cells that migrated through the 8μM pore membrane were fixed with methanol, nuclei stained with Propidium Iodide, and the nuclei were counted from digital images acquired using a microscope (model CKX41; Olympus America, Inc., Center Valley, P.A.)

    Techniques: Multiple Displacement Amplification, Incubation, Flow Cytometry, Cytometry, Software, Fluorescence, Activity Assay

    Effect of combined RQC or individual quercetin on breast cancer cell migration. Quiescent MDA-MB-231 and MDA-MB-435 cells were placed in the top of a transwell chamber with the bottom well containing: vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each, or 15μM quercetin in serum- and phenol red- free media. After 8h incubation, cells that migrated through the 8μM pore membrane were fixed, nuclei stained with Propidium Iodide and quantified. Percentage of migrated cells ± SEM for 15 microscopic fields/duplicate treatments for 3 independent experiments is presented. ( A) Average cell migration of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. (B) Average cell migration of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.

    Journal: PLoS ONE

    Article Title: Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    doi: 10.1371/journal.pone.0157251

    Figure Lengend Snippet: Effect of combined RQC or individual quercetin on breast cancer cell migration. Quiescent MDA-MB-231 and MDA-MB-435 cells were placed in the top of a transwell chamber with the bottom well containing: vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each, or 15μM quercetin in serum- and phenol red- free media. After 8h incubation, cells that migrated through the 8μM pore membrane were fixed, nuclei stained with Propidium Iodide and quantified. Percentage of migrated cells ± SEM for 15 microscopic fields/duplicate treatments for 3 independent experiments is presented. ( A) Average cell migration of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. (B) Average cell migration of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.

    Article Snippet: After 8h incubation, cells that migrated through the 8μM pore membrane were fixed with methanol, nuclei stained with Propidium Iodide, and the nuclei were counted from digital images acquired using a microscope (model CKX41; Olympus America, Inc., Center Valley, P.A.)

    Techniques: Migration, Multiple Displacement Amplification, Incubation, Staining

    The effect of oridonin nanosuspension and free oridonin on PANC-1 cell apoptosis was measured by annexin V-fluorescein isothiocyanate/propidium iodide staining. The early apoptotic cells stained by annexin-V-fluorescein isothiocyanate are located in the lower right quadrant. Abbreviation: ORI, oridonin.

    Journal: International Journal of Nanomedicine

    Article Title: Oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line

    doi: 10.2147/IJN.S29483

    Figure Lengend Snippet: The effect of oridonin nanosuspension and free oridonin on PANC-1 cell apoptosis was measured by annexin V-fluorescein isothiocyanate/propidium iodide staining. The early apoptotic cells stained by annexin-V-fluorescein isothiocyanate are located in the lower right quadrant. Abbreviation: ORI, oridonin.

    Article Snippet: Propidium iodide (PI), ribonuclease A, and annexin V-FITC were purchased from KeyGen Biotechnology ( Nanjing, China).

    Techniques: Staining

    Oridonin nanosuspension-induced and free oridonin-induced morphologic changes of PANC-1 cells. ( A ) Cellular morphology was examined in the presence of different doses of oridonin nanosuspension and free oridonin. Nuclear morphology was determined using ( B ) propidium iodide staining and ( C ) Hoechst 33342 staining. Abbreviation: ORI, oridonin.

    Journal: International Journal of Nanomedicine

    Article Title: Oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line

    doi: 10.2147/IJN.S29483

    Figure Lengend Snippet: Oridonin nanosuspension-induced and free oridonin-induced morphologic changes of PANC-1 cells. ( A ) Cellular morphology was examined in the presence of different doses of oridonin nanosuspension and free oridonin. Nuclear morphology was determined using ( B ) propidium iodide staining and ( C ) Hoechst 33342 staining. Abbreviation: ORI, oridonin.

    Article Snippet: Propidium iodide (PI), ribonuclease A, and annexin V-FITC were purchased from KeyGen Biotechnology ( Nanjing, China).

    Techniques: Staining

    SB225002 induces cell death in ALL cell lines. Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of (A) B-ALL and T-ALL cell lines. (B) Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. (C) Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. (D) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.

    Journal: PLoS ONE

    Article Title: SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

    doi: 10.1371/journal.pone.0134783

    Figure Lengend Snippet: SB225002 induces cell death in ALL cell lines. Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of (A) B-ALL and T-ALL cell lines. (B) Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. (C) Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. (D) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.

    Article Snippet: After 24 h of treatment, cells were centrifuged and fixed in cold 70% ethanol, washed twice with PBS and stained with 1 ml of propidium iodide solution (50 μg/ml propidium iodide, 3.8 mM sodium citrate in PBS) supplemented with 50 μl RNase A (50 mg/ml) for 1 h at room temperature and analyzed with a FACSCanto cell cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

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

    SB225002 induced cell death in ALL is mediated at least in part by the upregulation of GLIPR1 . (A) Relative proliferation of REH, RS4;11, Jurkat and TALL-1 cells upon sh.RNA knockdown of GLIPR1 (sh.GLIPR1) in comparison to control Scramble (sh.Scr). Number of viable cells were counted by flow cytometry and normalized to time-point zero. (B) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) scramble or GLIPR1 -knockdown cells treated with SB225002 [5 μM]. (C) Effect of SB225002 [5 μM or 10 μM] treatment on the proliferation of GLIPR1 -knockdown (sh. GLIPR1 ) versus control (sh.Scramble) B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cell lines. B-ALL cells were treated with SB225002 [10 μM] and T-ALL with SB225002 [5 μM]. Treatment control was DMSO 0.1%. Cells were incubated for 48 h. S = scramble transfection control; G-KD = cells infected with GLIPR1 -shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.

    Journal: PLoS ONE

    Article Title: SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

    doi: 10.1371/journal.pone.0134783

    Figure Lengend Snippet: SB225002 induced cell death in ALL is mediated at least in part by the upregulation of GLIPR1 . (A) Relative proliferation of REH, RS4;11, Jurkat and TALL-1 cells upon sh.RNA knockdown of GLIPR1 (sh.GLIPR1) in comparison to control Scramble (sh.Scr). Number of viable cells were counted by flow cytometry and normalized to time-point zero. (B) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) scramble or GLIPR1 -knockdown cells treated with SB225002 [5 μM]. (C) Effect of SB225002 [5 μM or 10 μM] treatment on the proliferation of GLIPR1 -knockdown (sh. GLIPR1 ) versus control (sh.Scramble) B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cell lines. B-ALL cells were treated with SB225002 [10 μM] and T-ALL with SB225002 [5 μM]. Treatment control was DMSO 0.1%. Cells were incubated for 48 h. S = scramble transfection control; G-KD = cells infected with GLIPR1 -shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.

    Article Snippet: After 24 h of treatment, cells were centrifuged and fixed in cold 70% ethanol, washed twice with PBS and stained with 1 ml of propidium iodide solution (50 μg/ml propidium iodide, 3.8 mM sodium citrate in PBS) supplemented with 50 μl RNase A (50 mg/ml) for 1 h at room temperature and analyzed with a FACSCanto cell cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Flow Cytometry, Cytometry, Incubation, Transfection, Infection, shRNA, Two Tailed Test