Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: HDL induces vasodilation in aortae from rats and mice in an eNOS-dependent manner. (a) Thoracic aortic rings from WKY rats were precontracted with PE (1 × 106 mol/l, arrows), and direct relaxation responses to HDL (0.5 mg/ml) or HDL and L-NAME (50 μmol/l) were evaluated. Shown are representative tracings from one experiment of 16. (b) Cumulative findings (mean ± SEM) for maximal relaxation in response to 0.5 mg/ml HDL in the presence of L-NAME (50 μmol/l), SKF-525A ((SKF, 50 μmol/l), or indomethacin (Indo, 10 μmol/l) (n = 6 each). *P < 0.01 vs. HDL. (c) HUVECs loaded with DAF-2DA were stimulated with 0.5 mg/ml HDL in the absence or presence of L-NAME. Cells were fixed and fluorescence was evaluated under a fluorescence microscope. Shown are representative results (n = 5). (d) Dose response of the vasodilatory effect of HDL (n = 3). (e) Thoracic aortic rings from WT 129/C57BL/6 mice and eNOS–/– mice were precontracted with PE, and direct relaxation responses to HDL (0.5 mg/ml) were measured. Shown are representative tracings from one experiment of six.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Fluorescence, Microscopy

Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: HDL induces NO release and vasodilation via Akt-mediated eNOS phosphorylation in endothelial cells and in aortic segments. (a) Left panel: [32P]orthophosphate-labeled HUVECs were stimulated with HDL (0.5 mg/ml) in the presence or absence of LY294002 (10 μmol/l). Immunoprecipitated eNOS (ip) was analyzed by autoradiography (n = 3), and amounts of immunoprecipitated protein were detected by Western blotting. Phosphorylation of Akt at Ser473 was determined in cell lysates with a phosphospecific antibody (n = 5). Right panel: Time-dependence of HDL-induced eNOS and Akt phosphorylation at Ser1177 and Ser473, respectively, as analyzed by densitometry (n = 3). (b) Following precontraction of thoracic aortic rings from WKY rats with PE (1 × 10–6 mol/l, arrows), direct relaxation responses to HDL (0.5 mg/ml) in the absence or presence of LY294002 (10 μmol/l) were evaluated. Shown are original tracings from one experiment of eight. (c) Aortic segments perfused with 0.5 mg/ml HDL were fixed and immunostained for phospho-Ser1177-eNOS. Arrows indicate phospho-eNOS staining in the endothelial lining (original magnification, ×200). (d) Fura2-AM–loaded HUVECs were stimulated with 1 mg/ml HDL in the presence or absence of BAPTA-2AM (20 μmol/l) or Ni2+ (5 mM). [Ca2+]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (e) HUVECs loaded with DAF-2DA and preincubated with BAPTA-2AM (20 μmol/l) or Ni2+ (5 mmol/l) were stimulated with HDL (1 mg/ml) and observed under a fluorescence microscope. Shown are representative results for one experiment of three.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Labeling, Immunoprecipitation, Autoradiography, Western Blot, Staining, Fluorescence, Spectroscopy, Microscopy

Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: Lysophospholipid signaling mediates HDL-, SPC-, S1P-, and LSF-induced Akt and eNOS phosphorylation as well as [Ca2+]i increase. (a) HUVECs were stimulated with HDL (0.5 mg/ml) or with 10 μmol/l each (left) or 0.5–5 μmol/l each (right) SPC, S1P, and LSF, with or without preincubation with 100 ng/ml PTX for 16 hours. “PTX control” indicates PTX treatment alone. Cell lysates were analyzed for phospho–Ser473-Akt (p-Ser473-Akt) and phospho–Ser1177-eNOS by Western blotting. Loading controls for total eNOS and total Akt content are shown. All results are representative of one experiment of three. (b) HUVECs loaded with DAF-2DA were stimulated with SPC, S1P, and LSF (10 μmol/l each) and observed under a fluorescence microscope. Shown are representative results for one experiment of three. (c) Fura2-AM–loaded HUVECs were stimulated with SPC, S1P, and LSF (10 μmol/l each), and [Ca2+]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (d) Concentration dependence of [Ca2+]i increase in HUVECs stimulated with SPC, S1P, and LSF measured as described in c.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Western Blot, Fluorescence, Microscopy, Spectroscopy, Concentration Assay

Journal: PLoS ONE

Article Title: DiGeorge Syndrome Gene tbx1 Functions through wnt11r to Regulate Heart Looping and Differentiation

doi: 10.1371/journal.pone.0058145

Figure Lengend Snippet: Dissected hearts from 72 hpf embryos showing the BA region (A, D) outlined in white, with the bidirectional arrowheads in black and red showing the length and width of BA, respectively. (B, E) The corresponding images showing staining for Alcama antibody (red) and DAF-2DA (green). (C, F) tbx1 −/− mutants at the same stage have absent eln2 expression. Black arrows indicate the BA. (G) Quantification of BA width in WT and tbx1−/− mutants, while (H) demonstrates that BA length is reduced in tbx1 −/− mutants; * p-value = 0.0045. N = 13 for all measurements. Scale bars: 25 µm.

Article Snippet: Labeling of bulbous arteriosus with DAF-2DA (VWR) was done as previously described .

Techniques: Staining, Expressing