Analysis results of rest quadrats. Co-occurrence patterns of ice-rafted dropstones and brachiopods in the Middle Permian Wandrawandian Siltstone of the southern Sydney Basin (southeastern Australia) and palaeoecological implications

Supplementary Material 2: Analysis results of rest quadrat

Original data collection. Co-occurrence patterns of ice-rafted dropstones and brachiopods in the Middle Permian Wandrawandian Siltstone of the southern Sydney Basin (southeastern Australia) and palaeoecological implications

Supplementary Material 1: Original data collectio

Table2_Role of ATG7-dependent non-autophagic pathway in angiogenesis.XLSX

ATG7, one of the core proteins of autophagy, plays an important role in various biological processes, including the regulation of autophagy. While clear that autophagy drives angiogenesis, the role of ATG7 in angiogenesis remains less defined. Several studies have linked ATG7 with angiogenesis, which has long been underappreciated. The knockdown of ATG7 gene in cerebrovascular development leads to angiogenesis defects. In addition, specific knockout of ATG7 in endothelial cells results in abnormal development of neovascularization. Notably, the autophagy pathway is not necessary for ATG7 regulation of angiogenesis, while the ATG7-dependent non-autophagic pathway plays a critical role in the regulation of neovascularization. In order to gain a better understanding of the non-autophagic pathway-mediated biological functions of the autophagy-associated protein ATG7 and to bring attention to this expanding but understudied research area, this article reviews recent developments in the ATG7-dependent non-autophagic pathways regulating angiogenesis.</p

Table1_Role of ATG7-dependent non-autophagic pathway in angiogenesis.XLSX

ATG7, one of the core proteins of autophagy, plays an important role in various biological processes, including the regulation of autophagy. While clear that autophagy drives angiogenesis, the role of ATG7 in angiogenesis remains less defined. Several studies have linked ATG7 with angiogenesis, which has long been underappreciated. The knockdown of ATG7 gene in cerebrovascular development leads to angiogenesis defects. In addition, specific knockout of ATG7 in endothelial cells results in abnormal development of neovascularization. Notably, the autophagy pathway is not necessary for ATG7 regulation of angiogenesis, while the ATG7-dependent non-autophagic pathway plays a critical role in the regulation of neovascularization. In order to gain a better understanding of the non-autophagic pathway-mediated biological functions of the autophagy-associated protein ATG7 and to bring attention to this expanding but understudied research area, this article reviews recent developments in the ATG7-dependent non-autophagic pathways regulating angiogenesis.</p

<i>Vn^−/−</i> mice impaired vascular permeability.

<p>(A). Whole-mount FITC-dextran angiograms in the WT and the <i>Vn^−/−</i> mice at day 7 in a mouse hindlimb ischemia model. Dextran leakage was more marked in the wild-type mice (upper panels, arrows) than in the <i>Vn^−/−</i> mice in ischemic gastrocnemius muscles. Capillaries were visualized by red Cy3-conjugated PECAM-1 antibodies. (B). Fluorescent intensities of cross-sections of non-ischemic (R2) and ischemic muscles (R1) were measured, and the R1/R2 ratio was used to assess the level of extravasation of FITC-dextran. Permeability was reduced in the gastrocnemius muscles in the <i>Vn^−/−</i> mice compared to the WT mice (n = 6, <i>p</i><0.05). (C). Electron microscopy of capillaries in the ischemic gastrocnemius muscles showed a diffuse and irregular basement membrane without distinct boundaries in WT mice in contrast to those of the <i>Vn^−/−</i> mice (upper panel, scale in 2 µm; lower panel is higher magnification. (D). The miles assay was performed with 50 ng/mL of VEGF and saline in the right and left ears, respectively, in the WT and <i>Vn^−/−</i> mice. Evan's blue dye extravasation was quantified with a spectrophotometer. The results are expressed as the mean ± SEM. * p<0.05. (n = 6 for each strain).</p

<i>Vn^−/−</i> mice reduced inflammatory cells infiltration.

<p>(A) Recruitment of macrophages in response to ischemia as determined by the Mac-3 antibody. Representative images of macrophages in nonischemic and ischemic muscles in the WT and <i>Vn^−/−</i> mice on day 7 after femoral artery ligation are shown. (B) The number of Mac3-positive cells was quantified. Data are the mean ± SEM from 10 fields per section (3 sections/mouse and n = 6 for each strain). *p<0.05. (C) The number of thioglycollate-elicited mouse peritoneal macrophages was quantified. The results are expressed as the means ± SEM. * p<0.05. (n = 6 for each strain). (D) The number of CD45-FITC- and PI- positive cells in peripheral blood was analyzed by flow cytometry after hindlimb ischemia by staining with CD45-FITC and PI (Propidium iodide). The results are expressed as the means ± SEM. * p>0.05. (n = 6 for each strain). (E). Transendthelial migration of murine mononuclear cells with TNFα of ECs. Murine aortic ECs were grown to confluency on cell culture inserts. ECs were then treated with 20 ng/mL TNFα for 4 hrs. Murine mononuclear cells were added on the endothelial layer for 4 hrs. Migrated cells were stained and measured by OD 450 nm according to the assay instructions. The results are expressed as the mean ± SEM. *p<0.05 vs. WT; **p<0.05 vs. control. Data shown are representative of three independent experiments.</p

Increased multimerization of VN in the tissue after ischemia.

<p>(A) and (B). RT-PCR and western blot analysis of VN from the gastrocnemius muscles indicated that ischemia induced VN expression in the WT mice (n = 6). R: Right leg; L: Left leg, NI: Nonischemic; I: Ischemic. (C). An equal amount of gastrocnemius muscle lysates (each 20 µg) were subjected to SDS-PAGE under nonreducing and reducing conditions and were analyzed by immunoblotting. The mobility of molecular weight standards for SDS-PAGE is indicated. Data are presented as fold changes (n = 9; *p<0.05 vs. control).</p

Mult VN promotes VE-cadherin internalization.

<p>(A) HUVECs were treated with mult VN (10 µg/mL). Confocal microscopy showed continuous and organized VE-cadherin staining, which is normally observed in ECs at cell-cell junctions (A-a). VN stimulation resulted in a disorganized pattern of VE-cadherin distribution at the membrane and led to intracellular accumulation of VE-cadherin (A-b); (B). To further determine whether cell surface-derived VE-cadherin was internalized into intracellular compartments, cells were prepared without acid washing and analyzed using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037195#s4" target="_blank"><i>Methods</i></a>. A time-dependent internalization of VE-cadherin was observed (upper panels, B-a-d). Pretreatment with LM 609 (LM) and su6656 (su) prevented VN-mediated VE-cadherin internalization (B-e, f, lower panels). VE-cadherin internalization was not observed after treatment with mono VN (10 µg/mL)(B-h). VEGF (50 ng/mL) as a positive control (B-g). (C). Cells were prepared by acid washing and analyzed using confocal microscopy and cell surface VE-antibody was removed with a low pH acid wash. VN treatment increased the intracellular accumulation of VE-cadherin internalized from the cell surface (C-b, c) relative to no treatment (C-a). Nuclei are stained with DAPI. The scale bars represent 10 µm. Arrows: cell surface; Arrowhead: intracellular accumulation. (D). HUVEC monolayers were treated with VN at the indicated dose. Cells were lysed using RIPA buffer for western blotting analysis of total VE-cadherin. (E). Cells were treated with trypsin/EDTA, pelleted and lysed with RIPA for western blotting analysis of intracellular VE-cadherin. (F). HUVECs were transfected with Src siRNA or scrambled siRNA (con siRNA). The efficiency of Src protein expression knock-down was analyzed using immunoblot (left panel). Cells were treated with trypsin/EDTA, pelleted and lysed with RIPA for western blotting analysis of intracellular VE-cadherin (right panel). β-actin was used as a loading control.</p

Additional file 1 of RAGE displays sex-specific differences in obesity-induced adipose tissue insulin resistance

Additional file 1: Figure S1. Female RAGE deficiency improved glucose and insulin tolerance in normal diet mice. (A) Glucose tolerance tests (GTT) and Area under the curve (AUC) in each group. n = 6 per group. *p < 0.05 vs. female RAGE−/−-HFD-F mice; #p < 0.05 vs. female RAGE−/−-ND-F mice. (B) Insulin tolerance tests (ITT) and AUC in each group. n = 6 per group. *p < 0.05 vs. female RAGE−/−-HFD-F mice; #p < 0.05 vs. female RAGE−/−-ND-F mice. ND; normal diet. All group data are shown as mean ± SEM

Additional file 2 of RAGE displays sex-specific differences in obesity-induced adipose tissue insulin resistance

Additional file 2: Table S1. Sequences of primers used in the study