Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.

doi: 10.1016/j.matbio.2013.12.011

Figure Lengend Snippet: Fig. 2. Decorin stimulates formation of a Vps34/Beclin 1 complex while simulta- neously inhibiting formation of the Bcl-2/Beclin 1 complex. [A–C] Representative co-immunoprecipitation experiments performed in HUVEC with Beclin 1 [A], Vps34 [B], or VEGFR2 [C] immunoprecipitates following stimulation with 200 nM decorin for 6 h. All immunoprecipitates were executed with 5 μg of appropriate primary antibody. Notably, for Beclin 1 immunodetection [A,B], membranes were incubated with a mouse anti-rabbit light chain antibody following primary antibody incubation that effectively switches the antibody isotype from rabbit to mouse and thereby precludes detection of the heavy IgGchain in this region from immunoprecipitates. Allexperiments wererepeated at least three independent times with similar results.

Article Snippet: The monoclonal rabbit antibodies against human VEGFR2, Phospho-VEGFR2 (detecting Tyr1175), Akt, Phospho-Akt (at residue Thr308), mTOR, Phospho-mTOR (at residue Ser2448), p70S6K, Phospho-p70S6K (at residue Thr389), PI3 kinase class III (Vps34), Phospho-AMPKα (at residue Thr172), AMPKα mouse anti-rabbit light chain IgG, and GAPDH were procured from Cell Signaling.

Techniques: Immunoprecipitation, Immunodetection, Incubation

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.

doi: 10.1016/j.matbio.2013.12.011

Figure Lengend Snippet: Fig. 5. Decorin promotes activation of a pro-autophagic pathway via AMPKα phosphorylation. [A] Representative immunoblot interrogating P-AMPKα (Thr172) in response to decorin (200 nM) at the indicated time points [B,C] Representative immunoblots depicting RNAi-mediated silencing of VEGFR2 (siVEGFR2, 80 pM) relative to siScr control (20 pM) in HUVEC stim- ulated with 200 nM decorin for 1 h [B] and corresponding quantification [C]. [D,E] Gene expression analysis via qPCR of BECN1 [D] and MAPLC3A [E] following pre-treatment with Com- pound C (30 μM, 30 min) followed by a challenge with decorin (200 nM, 4 h). Data were normalized to the endogenous housekeeping gene, ACTB. [F,G] Representative immunoblots of Beclin 1 [F] following pre-incubation with Compound C (30 μM, 30 min) followed by decorin (200 nM, 4 h) and quantified [G] after normalization to GAPDH. [H] Immunofluorescence imaging of LC3-positive autophagosome formation ensuing treatment with either decorin alone (200 nM, 3 h) or in combination with Compound C (30 μM, 30 min pre-treatment) in HUVEC. All images were procured with the same gain, intensity, and exposure. Nuclei were visualized with DAPI. [I] Quantification of the number of autophagosomes per cell in HUVEC treated with decorin only (200 nM, 3 h) or pre-treated with Compound C (30 μM, 30 min). Fold changes ± SEM have been reported for gene expression analyses [D,E] and were calculated in accordance with the ΔΔCt method (please consult Section 4.6 for more information). All experiments reported were repeated at least three independent times. ***p b 0.001; **p b 0.01.

Article Snippet: The monoclonal rabbit antibodies against human VEGFR2, Phospho-VEGFR2 (detecting Tyr1175), Akt, Phospho-Akt (at residue Thr308), mTOR, Phospho-mTOR (at residue Ser2448), p70S6K, Phospho-p70S6K (at residue Thr389), PI3 kinase class III (Vps34), Phospho-AMPKα (at residue Thr172), AMPKα mouse anti-rabbit light chain IgG, and GAPDH were procured from Cell Signaling.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Gene Expression, Incubation, Imaging

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.

doi: 10.1016/j.matbio.2013.12.011

Figure Lengend Snippet: Fig. 6. Working model depicting the proposed mechanism through which decorin induces endothelial cell autophagy via differential modulation of anti- and pro-autophagic signal- ing cascades downstream of VEGFR2. Decorin functions as a soluble pro-autophagic signal via binding cell surface localized VEGFR2 at the endothelial cell membrane for potent and sustained activation of AMPKα. Concurrently, decorin attenuates the anti-autophagic arm (Akt/mTOR/p70S6K). Moreover, decorin promotes the differential assembly of Vps34/ Beclin 1 positive complexes by overriding the putatively repressive association of Beclin 1 with Bcl-2. Collectively, modulation of these signaling cascades provides mechanistic in- sights into decorin evoked endothelial cell autophagy.

Article Snippet: The monoclonal rabbit antibodies against human VEGFR2, Phospho-VEGFR2 (detecting Tyr1175), Akt, Phospho-Akt (at residue Thr308), mTOR, Phospho-mTOR (at residue Ser2448), p70S6K, Phospho-p70S6K (at residue Thr389), PI3 kinase class III (Vps34), Phospho-AMPKα (at residue Thr172), AMPKα mouse anti-rabbit light chain IgG, and GAPDH were procured from Cell Signaling.

Techniques: Binding Assay, Membrane, Activation Assay

Journal: Cell reports

Article Title: Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

doi: 10.1016/j.celrep.2019.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Polyclonal anti-Flk1-1 , Acris Antibodies GmbH , Cat# AP02618PU-S; RRID: AB_1624459.

Techniques: Control, Recombinant, Clinical Proteomics, Modification, Membrane, Sterility, Reverse Transcription, DNA Methylation Assay, Flow Cytometry, Methylation, Software, Imaging, Cell Culture