Journal: The Journal of Biological Chemistry

Article Title: Group V secreted phospholipase A 2 plays a protective role against aortic dissection

doi: 10.1074/jbc.RA120.013753

Figure Lengend Snippet: Expression of sPLA2s in the aortas of mice and humans. A, expression of sPLA2 mRNAs relative to Gapdh in the aorta of WT C57BL/6 mice (n = 4). B, expression of Pla2g5 in various tissues of WT mice (n = 4). C, immunohistochemistry of sPLA2-V in the aorta of Pla2g5+/+ and Pla2g5−/− mice. Arrows, positive staining of sPLA2-V. Scale bars, 100 μm. D, flow cytometry of CD31-positive/negative cells from WT mouse aorta. E, expression of sPLA2 mRNAs of CD31 positive/negative cells from WT mouse aorta (n = 3). F, schematic procedure of AT-II infusion into mice using subcutaneously implanted osmotic pumps. G, time course of the expression of sPLA2 mRNAs in WT mouse aorta after AT-II infusion (n = 4). H, immunofluorescence of sPLA2-V (green) and CD31 (magenta) with DAPI (blue) in the aorta of Pla2g5+/+ and Pla2g5−/− mice with or without AT-II infusion for 48 h. The rightmost panels represent the staining of Pla2g5+/+ aorta after AT-II infusion, following perfusion with heparinized saline through the left ventricle before extraction. Arrows indicate positive staining of sPLA2-V. Scale bars, 20 μm. I, expression of PLA2G5 in nondissecting or dissecting site of human aorta (n = 6). **, p < 0.01 by unpaired t test. J, a representative section of human aorta stained with HE and immunohistochemistry of the serial sections with control IgG (Ctrl), anti-CD31 antibody, or anti-sPLA2-V antibody. A boxed area is magnified on the right. Arrows, positive staining. Scale bars, 100 μm. Data are presented as mean ± S.E. (error bars) of the indicated number (n) of biological replicates.

Article Snippet: For flow cytometry analysis, the cells were stained with phycoerythrin-conjugated anti-mouse CD31 antibody (130-102-608, Miltenyi Biotec) and analyzed using a cell analyzer (EC800, Sony Biotechnology).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Group V secreted phospholipase A 2 plays a protective role against aortic dissection

doi: 10.1074/jbc.RA120.013753

Figure Lengend Snippet: Decreased LOX expression and function in aorta of AT-II–infused Pla2g5−/− mice. A, mRNA expression of LOX family members, vascular remodeling markers, and pro-inflammatory cytokines in the aortas of Pla2g5+/+ and Pla2g5−/− mice with or without AT-II infusion for 48 h (n = 4–5). B, time course of Lox mRNA expression in the aortas of Pla2g5+/+ and Pla2g5−/− mice after AT-II infusion (n = 3–8). Shown are immunoblotting of LOX protein, with GAPDH as an internal control (C), densitometric analysis of mature LOX protein relative to GAPDH (n = 3) (D), LOX activity (n = 4–5) (E), and insoluble/soluble collagen ratio (n = 3–4) (F) in the aortas of Pla2g5+/+ and Pla2g5−/− mice with or without AT-II infusion for 48 h. *, p < 0.05; **, p < 0.01; ns, not significant by two-way ANOVA with Tukey's multiple-comparison test. Data are presented as mean ± S.E. (error bars) of the indicated number (n) of biological replicates (A, B, D, E, and F). G, immunofluorescence of aortas of Pla2g5+/+ and Pla2g5−/− mice with or without AT-II infusion for 48 h using control IgG (Ctrl), anti-LOX antibody (green), and anti-CD31 antibody (magenta) with DAPI (blue). Scale bars, 20 μm. H, correlation of PLA2G5 and LOX expression in human aorta (n = 12). Pearson correlation (r value) and statistical significance (p value) are indicated.

Article Snippet: For flow cytometry analysis, the cells were stained with phycoerythrin-conjugated anti-mouse CD31 antibody (130-102-608, Miltenyi Biotec) and analyzed using a cell analyzer (EC800, Sony Biotechnology).

Techniques: Expressing, Western Blot, Activity Assay, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Group V secreted phospholipase A 2 plays a protective role against aortic dissection

doi: 10.1074/jbc.RA120.013753

Figure Lengend Snippet: Increased aortic dissection in EC-specific Pla2g5-null mice. A, schematic procedure for generation of EC-specific Pla2g5-null mice. B, expression of Pla2g5 mRNA relative to Gapdh in the aorta and heart of control (f/f) and EC-specific Pla2g5-null (f/f Tie2-Cre) mice (n = 4). C, representative thoracic aortas of control and EC-specific Pla2g5-null mice after 7 days of AT-II infusion. Arrows indicate aortic dissection with intramural hematoma. Scale bars, 1 mm. D, incidence of thoracic aortic dissection or rupture in control (n = 14) and EC-specific Pla2g5-null (n = 17) mice within 7 days of AT-II infusion. E, mRNA expression of Lox, vascular remodeling markers (Mmp2, Acta2, and Tgfb1), and pro-inflammatory cytokine (Tnf) in the aortas of control and EC-specific Pla2g5-null mice with or without AT-II infusion for 48 h (n = 4–6). Shown are immunoblot analysis of LOX protein, with GAPDH as an internal control (F), densitometric analysis of mature LOX protein relative to GAPDH (n = 3) (G), and LOX activity (n = 4) (H) in the aortas of control and EC-specific Pla2g5-null mice with or without AT-II infusion for 48 h. I, immunofluorescence of aortas of control and EC-specific Pla2g5-null mice with or without AT-II infusion for 48 h using control IgG (Ctrl) or anti-LOX antibody (green) and anti-CD31 antibody (magenta) with DAPI (blue). Scale bars, 20 μm. *, p < 0.05; **, p < 0.01; ns, not significant by two-way ANOVA (B, E, G, and H) and by Fisher's exact test (D). Data are presented as mean ± S.E. (error bars) of the indicated number (n) of biological replicates.

Article Snippet: For flow cytometry analysis, the cells were stained with phycoerythrin-conjugated anti-mouse CD31 antibody (130-102-608, Miltenyi Biotec) and analyzed using a cell analyzer (EC800, Sony Biotechnology).

Techniques: Dissection, Expressing, Western Blot, Activity Assay, Immunofluorescence

Journal: Bone Marrow Transplantation

Article Title: Brief ex vivo Fas-ligand incubation attenuates GvHD without compromising stem cell graft performance

doi: 10.1038/s41409-020-0941-2

Figure Lengend Snippet: a – h MPBC graft characterization following FasL treatment. Percentage of annexin V positive a CD3 + and b CD34 + cells. c Percentage of CD3 + and d CD34 + cells per total CD45 + population. HSPCs subpopulations; e Immature (CD34 + CD38 low ), f Multipotent progenitors (CD45RA − CD90 − ) and g self-renewing hematopoietic stem cells (CD45RA − CD90 + ). h Colony-forming units (CFU) profile of: erythroid progenitor cells (CFU-E and BFU-E), granulocyte-macrophage progenitor cells (CFU-GM) and multipotential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-GEMM). Engraftment, differentiation and CFU potential as detected in the BM of γ-irradiated (2.75 Gy) NSG mice, 4 weeks post transplantation of 1 × 10 5 human CD34 + cells: i human leukocytes (hCD45 + ) j immature hCD34 + CD38 low progenitors and k human leukocytes subpopulations: B (hCD19 + ), Myelo-monocytic (hCD33 + and CD14 + CD16 − ), NK (hCD56 + CD16 − ), HSPCs (CD34 + ) cells and l number of human colony-forming cells in the mice BM. Data presented as ( a – h ) mean+SD or ( i – l ) individual mice and median. ( a – d ) n = 25 or ( e – h ) n = 3 individual MPBCs donations and ( i – l ) show one representative study out of two, n = 7 (MPBC-CD34) and n = 8 (MPBC + FasL-CD34) female mice per group transplanted with cells from one individual MPBC donation. Statistical analysis preformed using ( a – d ) ANOVA parametric test, ( e – h ) paired T-test or ( i – l ) Mann–Whitney test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: anti- mouse CD45 (130-102-412), human CD45 (130-098-143, 130-098-151, 130-098-141, 130-098-148, 130-104-566, 130-110-637), CD34 (130-081-002, 130-090-954), CD90 (130-099-289), CD3 (130-109-460, 130-109-466), CD4 (130-110-680), CD8 (130-109-454), CCR7 (130-108-309), CD45RA (130-108-784, 130-110-637), LFA1 (130-105-437), CD95 (130-104-232), CXCR3 (130-101-378), CCR6 (130-100-377), CD25 (130-109-021), CD33 (130-111-021), CD19 (130-113-642, 130-110-249), CD27 (130-099-499), CD38 (130-108-862), HLA-DR (130-104-825), IL17 (130-100-077), IFN-γ (130-097-944) and FoxP3 (130-098-119) all purchased from Miltenyi Biotech (Bergisch Gladbach, Germany); matched isotype controls were used as negative control.

Techniques: Irradiation, Transplantation Assay, MANN-WHITNEY