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