Journal: The FASEB Journal

Article Title: Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis

doi: 10.1096/fj.201903082r

Figure Lengend Snippet: FIGURE 2 Hypoxia decreases the percentage of endothelial cells in the S-phase of the cell cycle. Exponentially growing asynchronous HUVEC were exposed to 21% oxygen (N) or 1% oxygen (Hx) and the cells were pulse labeled with EdU to quantify the percentage of S-phase cells by microscopy (A and B) or flow cytometry (C and D), respectively. A, A Representative microscopy image of each experimental condition (24 hours or 48 hours N or Hx) is shown. EdU+ nuclei are shown in magenta and nuclei were visualized by DAPI staining. Bar: 100 μm. B, Percentage of EdU+ cells/field in N (blue circles) or Hx (red triangles) in 3-5 independent experiments (10 fields were quantified per experimental condition and each symbol corresponds to the mean value of one experiment). Statistical significance was determined by one-way ANOVA using Tukey’s post-test (*P < .05, **P < .01). C, Representative plot of EdU-Alexa 647 vs DNA content by propidium iodide staining (PI) of each experimental condition (72 hours N or Hx) is shown. Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle is shown inside the corresponding gating regions (magenta lines). D, Percentage of cells in G0/G1, S, and G2/M phases quantified by FACS in N (blue circles) or Hx (red triangles). Symbols correspond to the value of independent experiments and bars represent the mean of 4-5 independent experiments. Statistical significance was determined by two-way ANOVA using Sidak’s post-test (**P < .01, ***P < .001, ****P < .0001). E, Cell proliferation curves in N (blue circles) or Hx (red triangles). Cell number was quantified by flow cytometry using perfect count microspheres. Each point represents the mean of three independent experiments. Statistical significance was determined by two-way ANOVA using Sidak’s post-test (*P < .05, **P < .01). F, Linear regression analysis of proliferation curves. The doubling time was significantly reduced in hypoxia compared to normoxia (Single sample paired Student’s t-test, P < .05)

Article Snippet: Cells were washed with 1 mL of 0.1% Tween-20 in PBS, centrifuged and resuspended in 200 μL of PBS containing 250 μg/ mL of RNase and 10 μg/mL of propidium iodide (PI) (Cell Signaling Technology, 4087S) or 1 μg/mL of 4′,6-diamidino2-phenylindol (DAPI Molecular Probes, D1306) followed by an incubation at RT protected from light.

Techniques: Labeling, Microscopy, Flow Cytometry, Staining

Journal: The FASEB Journal

Article Title: Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis

doi: 10.1096/fj.201903082r

Figure Lengend Snippet: FIGURE 3 HIF mutants stabilized in normoxia decrease the percentage of endothelial cells in the S-phase of the cell cycle. Exponentially growing asynchronous HUVEC were infected with control lentivirus (pRRL), lentivirus for the expression of EPAS1 or HIF1A mutants stabilized in normoxia (pRRL-EPAS1PP, pRRL-HIF1αPP), or lentivirus for the expression of double mutants whose bHLH domain has also been mutated (pRRL-EPAS1PPbHLH*, pRRL-HIF1αPPbHLH*). Transduced HUVEC were pulse labeled with EdU to quantify the percentage of S-phase cells by microscopy (A-B) or flow cytometry (C-D) 48 hours after infection. A, A representative microscopy image of each experimental condition (N pRRL, Hx pRRL, pRRL-EPAS1PP, pRRL-HIF1αPP, pRRL-EPAS1PPbHLH*, pRRL-HIF1αPPbHLH*) 48 hours after infection is shown. EdU+ nuclei are shown in magenta and nuclei were visualized by DAPI staining. Bar: 100 μm. B, Percentage of EdU+ cells/field in 3-4 independent experiments (10 fields were quantified per experimental condition and each symbol corresponds to the mean value of one experiment) 48 hours after infection. Statistical significance was determined by one-way ANOVA using Tukey’s post-test (*P < .05, **P < .01, ***P < .001). C, A representative plot of EdU-Alexa 647 vs DNA content propidium iodide (PI) of each experimental condition 48 hours after infection is shown. Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle is shown inside the corresponding gating regions (magenta lines). D, Percentage of cells in G0/G1, S, and G2/M phases quantified by FACS for each experimental condition 48 hours after infection. Symbols correspond to the value of independent experiments and bars represent the mean of 3-4 independent experiments. Statistical significance was determined by two-way ANOVA using Tukey’s post-test (*P < .05, **P < .01)

Article Snippet: Cells were washed with 1 mL of 0.1% Tween-20 in PBS, centrifuged and resuspended in 200 μL of PBS containing 250 μg/ mL of RNase and 10 μg/mL of propidium iodide (PI) (Cell Signaling Technology, 4087S) or 1 μg/mL of 4′,6-diamidino2-phenylindol (DAPI Molecular Probes, D1306) followed by an incubation at RT protected from light.

Techniques: Infection, Control, Expressing, Labeling, Microscopy, Flow Cytometry, Staining

Journal: The FASEB Journal

Article Title: Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis

doi: 10.1096/fj.201903082r

Figure Lengend Snippet: FIGURE 4 Hypoxia-Inducible Factors are required to withdraw HUVEC from S-phase entry in hypoxic conditions. Exponentially growing asynchronous HUVEC were transduced with lentivirus for the expression of shRNAs to specifically silence EPAS1 (pGIPZ-shEPAS1) or HIF1A (pGIPZ-shHIF1α) and a scramble, non-silencing shRNA, was used as control (pGIPZ-shScr) in N or Hx. A, Levels of EPAS1 and HIF1A mRNA were determined by qRT-PCR. The graph represents the ratio over scramble shSrc of four independent experiments. Statistical significance was determined by two-way ANOVA using Tukey’s post-test (****P < .0001). B, HIF1α and EPAS1 protein expression was analyzed by western- blot. C, Levels of ANGPTL4 and BNIP3 mRNA in transduced HUVEC were determined by qRT-PCR. The graph represents the ratio over N pGIPZ-shSrc of four independent experiments. Statistical significance was determined by one-way ANOVA using Tukey’s post-test (*P < .05). Transduced HUVEC were pulse labeled with EdU to quantify the percentage of S-phase cells by microscopy (D-E) or FACS (F-G) 72 hours after lentiviral infection. D, A representative microscopy image of each experimental condition (pGIPZ-shScr, pGIPZ-shEPAS1 and pGIPZ-shHIF1α) in N or Hx is shown. EdU+ nuclei are shown in magenta and nuclei were visualized by DAPI staining. Bar: 100 μm. E, Box plot of percentage EdU+ cells/field for each experimental condition. Each box represents the mean of two independent experiments (10 fields/experiment) Statistical significance was determined by one-way ANOVA using Tukey’s post-test (***P < .001). F, A representative plot of EdU-Alexa 647 vs DNA content propidium iodide (PI) for each experimental condition (pGIPZ-shScr, pGIPZ-shEPAS1 and pGIPZ-shHIF1α) in N or Hx conditions is shown. Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle is shown inside the corresponding gating regions (magenta lines). G, Percentage of cells in G0/G1, S, and G2/M phases quantified by FACS for each experimental condition 48 hours after infection. Symbols correspond to the value of independent experiments and bars represent the mean of four independent experiments. Statistical significance was determined by two-way ANOVA using Tukey’s post-test (**P < .01, ***P < .001, ****P < .0001)

Article Snippet: Cells were washed with 1 mL of 0.1% Tween-20 in PBS, centrifuged and resuspended in 200 μL of PBS containing 250 μg/ mL of RNase and 10 μg/mL of propidium iodide (PI) (Cell Signaling Technology, 4087S) or 1 μg/mL of 4′,6-diamidino2-phenylindol (DAPI Molecular Probes, D1306) followed by an incubation at RT protected from light.

Techniques: Transduction, Expressing, shRNA, Control, Quantitative RT-PCR, Western Blot, Labeling, Microscopy, Infection, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells

doi: 10.3892/etm.2015.2692

Figure Lengend Snippet: Effect of EESB on the HT-29 cell cycle. Cells were pretreated with various concentration of EESB for 1 h, followed by stimulation with 10 ng/ml IL-6 for 24 h. (A) Cells were stained with propidium iodide and analyzed by fluorescence-activated cell sorting. The proportion of DNA in the S phase was calculated using ModFit LT version 3.0 software. (B) Quantification of fluorescence-activated cell sorting analysis. The data shown are averages with standard deviation from 3 independent experiments. #P<0.05 vs. cells treated with IL-6 but not EESB. EESB, ethanol extract of Scutellaria barbata D. Don; IL-6, interleukin-6.

Article Snippet: The HT-29 cell cycle progression was determined through flow cytometric analysis using a propidium iodide (PI) staining cell cycle assay kit (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Concentration Assay, Staining, Fluorescence, FACS, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells

doi: 10.3892/etm.2015.2692

Figure Lengend Snippet: Effect of EESB on HT-29 cell apoptosis. Cells were pretreated with various concentration of EESB for 1 h, followed by stimulation with 10 ng/ml IL-6 for 24 h. (A) Cells were collected and stained with Annexin V/PI, followed by fluorescence-activated cell sorting analysis. Double-negative stained cells indicate the live cell population; Annexin V-positive/PI-negative stained cells and Annexin V/PI double-positive stained cells represent early and late apoptosis, respectively; Annexin V-negative and PI-positive stained cells show dead cells. (B) Quantification of fluorescence-activated cell sorting analysis. The data shown are averages with standard deviation from 3 independent experiments. #P<0.05 vs. cells treated with IL-6 but not EESB. EESB, ethanol extract of Scutellaria barbata D. Don; IL-6, interleukin-6; UL, upper left; UR, upper right; LR, lower right; LL, lower left; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Article Snippet: The HT-29 cell cycle progression was determined through flow cytometric analysis using a propidium iodide (PI) staining cell cycle assay kit (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Concentration Assay, Staining, Fluorescence, FACS, Standard Deviation

Journal: Respiratory Research

Article Title: Anti-inflammatory potential of PI3Kδ and JAK inhibitors in asthma patients

doi: 10.1186/s12931-016-0436-2

Figure Lengend Snippet: Effects of dexamethasone, PIK-294 and tofacinib on TCR-induced pSTAT5 activation in lymphocytes. Isolated blood T-cells from asthma patients ( n = 5) and healthy subjects ( n = 6) and BAL cells ( n = 4 asthma plus n = 3 healthy) were treated with 1000nM dexamethasone, PIK-294 or tofacinib for 1 h before TCR-stimulation for 4 h. Levels of phosphorylated STAT5 in CD3 + cells were quantified by flow cytometry. Data is presented as mean % pSTAT5 positive CD3 cells +/− standard deviation. Drug effects were assessed by 1-way ANOVA with a Dunnett’s multiple comparison test against the stimulated no drug control: * p < 0.05

Article Snippet: Cytotoxic effects of the drugs were assessed in TCR-stimulated PBMCs by Pierce LDH assay (Life Technologies, Paisley, UK) and propidium iodide flow cytometry assay (BD Bioscience, Oxford, UK); Additional file : Figure S1.

Techniques: Activation Assay, Isolation, Flow Cytometry, Standard Deviation

Journal: Respiratory Research

Article Title: Anti-inflammatory potential of PI3Kδ and JAK inhibitors in asthma patients

doi: 10.1186/s12931-016-0436-2

Figure Lengend Snippet: Expression of PI3Kδ in subepithelial and BAL T-cells from asthma patients and healthy subjects. PI3Kδ expression was examined in CD3 cells in subepithelial bronchial tissue and BAL by immunohistochemistry and flow cytometry, respectively. Representative images show PI3Kδ expression in bronchial tissue from healthy subjects ( a ) and asthma patients ( b ). Arrows and amplified images highlight example staining: Red for CD3 only; Blue for PI3Kδ only; Green for dual CD3 and PI3Kδ. Black scale bars represent 50 μm. Data is presented as ( c ) numbers of CD3 + PI3Kδ + cells per mm 2 subepithelium (Healthy n = 10; Asthma n = 9); ( d ) percentage of subepithelial CD3 cells expressing PI3Kδ (Healthy n = 8; Asthma n = 8); ( e ) percentage of BAL CD3 cells expressing PI3Kδ (Healthy n = 6; Asthma n = 9) and ( f ) PI3Kδ-dependent mean fluorescence intensity in dual labelled CD3 + PI3Kδ + BAL cells (Healthy n = 6; Asthma n = 9), with bar illustrating mean values. The percentage of subepithelial CD3 cells expressing PI3Kδ could not be calculated for n = 2 healthy and n = 1 asthma, due to lack of CD3 cells. Differences between healthy and asthma were assessed by T-test: * p < 0.05; ** p < 0.01

Article Snippet: Cytotoxic effects of the drugs were assessed in TCR-stimulated PBMCs by Pierce LDH assay (Life Technologies, Paisley, UK) and propidium iodide flow cytometry assay (BD Bioscience, Oxford, UK); Additional file : Figure S1.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry, Amplification, Staining, Fluorescence