Journal: Journal of vascular surgery

Article Title: RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

doi: 10.1016/j.jvs.2017.11.091

Figure Lengend Snippet: Angiotensin II (Ang II)-induced receptor activator of nuclear factor κB ligand (RANKL) expression in smooth muscle cells. Murine vascular smooth muscle cells (MOVAS cells) were stimulated with Ang II. Expression of RANKL messenger RNA (mRNA) and protein was quantified by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Forskolin (FSK) was used as a positive control. Ang II dose dependently induced Rankl mRNA (A) and RANKL protein expression (B). A time-course experiment showed increased Rankl mRNA (C) and RANKL protein (D) from 24 to 72 hours with a maximum at around 48 hours. Relative expression of Western blots was quantified using ImageJ. E and F, Increased RANKL expression through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway in smooth muscle cells. MOVAS cells were stimulated with 1 μM Ang II. JAK2 and STAT5 activities were assayed in Western blots. E, JAK2 and STAT5 phosphorylation was increased at 12 hours. F, Effect of TG101348, a selective inhibitor of JAK2, on Ang II-induced RANKL expression. TG101348 suppressed Ang II-induced RANKL expression in MOVAS cells. G-I, Analysis of the effects of Ang II and RANKL-neutralizing antibody on MOVAS cells. Cells were cultured in growth medium alone or media containing Ang II (1 μM), RANKL-neutralizing antibody (100 ng/mL), or both for 48 hours. G, Mitochondrial membrane potential of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. H, Proliferation of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. OD, optical density. I, Flow cytometric analysis of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. Cells were analyzed for the expression of tissue inhibitor of metalloproteinase 1 (TIMP1), transforming growth factor β (TGF-β), bone morphogenetic protein 4 (BMP4), matrix metalloproteinase (MMP) 9, and MMP2. Flow cytometric data are represented as the mean fluorescence intensity ± standard deviation of four replicates.

Article Snippet: Cells were resuspended in permeabilization buffer containing the following primary antibodies: anti-transforming growth factor β (TGF-β)-Alexa Fluor 700 (IC1835N; R&D Systems, Minneapolis, Minn), anti-bone morphogenetic protein 4 (BMP4)-Alexa Fluor 647 (sc-12721; Santa Cruz Biotechnology), anti-MMP-2-phycoerythrin (sc-13594; Santa Cruz Biotechnology), anti-tissue inhibitor of metalloproteinase 1 (TIMP1)-biotin (BAF980; R&D Systems), anti-RANKL-Alexa Fluor 405 (NB100–56593AF405; Novus Biologicals, Littleton, Colo), and anti-Mmp9- peridinin-chlorophyll (SAB5200309; Sigma-Aldrich).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control, Phospho-proteomics, Cell Culture, Membrane, Fluorescence, Standard Deviation

Journal: Journal of vascular surgery

Article Title: RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

doi: 10.1016/j.jvs.2017.11.091

Figure Lengend Snippet: Receptor activator of nuclear factor κB ligand (RANKL)-mediated stimulation of macrophages. RAW 264.7 macrophages were cultured with RANKL for 2 days, and matrix metalloproteinase 9 (MMP9) messenger RNA (mRNA) and protein expression was determined in the cell lysates by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. A, Mmp9 mRNA expression was dose dependently increased by RANKL stimulation. B, Representative Western blot and densitometric analysis of MMP9 protein expression in cell lysates of RAW264.7 cells treated with RANKL. Relative expression of Western blots was quantified using ImageJ. C-E, Analysis of the effects of angiotensin II (Ang II) and RANKL-neutralizing antibody on RAW 264.7 cells. Cells were cultured in growth medium alone or media containing Ang II (1 μM), RANKL-neutralizing antibody (100 ng/mL), or both for 48 hours. C, Mitochondrial membrane potential of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. D, Proliferation of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. OD, optical density. E, Flow cytometric analysis of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. Cells were analyzed for the expression of tissue inhibitor of metalloproteinase 1 (TIMP1), transforming growth factor β (TGF-β), bone morphogenetic protein 4 (BMP4), MMP9, MMP2, and RANKL. Flow cytometric data are represented as the mean fluorescence intensity ± standard deviation of four replicates.

Article Snippet: Cells were resuspended in permeabilization buffer containing the following primary antibodies: anti-transforming growth factor β (TGF-β)-Alexa Fluor 700 (IC1835N; R&D Systems, Minneapolis, Minn), anti-bone morphogenetic protein 4 (BMP4)-Alexa Fluor 647 (sc-12721; Santa Cruz Biotechnology), anti-MMP-2-phycoerythrin (sc-13594; Santa Cruz Biotechnology), anti-tissue inhibitor of metalloproteinase 1 (TIMP1)-biotin (BAF980; R&D Systems), anti-RANKL-Alexa Fluor 405 (NB100–56593AF405; Novus Biologicals, Littleton, Colo), and anti-Mmp9- peridinin-chlorophyll (SAB5200309; Sigma-Aldrich).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Membrane, Fluorescence, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells

doi: 10.3390/ijms242316851

Figure Lengend Snippet: Conditioned media from G17-stimulated melanoma cells exhibit upregulation of MMP-2 and downregulation of TIMP-3 expression. Representative demonstration of the confirmation of proteomic data using Western blot analysis, with prosaposin serving as a benchmark for gastrin responsiveness ( A ). Mean and standard deviation (±SD) of the densitometric analysis results from three independent experiments ( B ). Statistical difference (* p < 0.05) between treated and untreated control groups (considered as 100%) are indicated as follows: prosaposin, TIMP-3 and TIMP-1 levels in G361 cells and MMP2 expressions in the SK-MEL-2 cell line.

Article Snippet: Antibodies against MMP-2, TIMP-3 (R&D Systems Abingdon, UK; AF902 and MAB973, respectively), TIMP-1, TIMP-2, prosaposin, and MMP-1 (R&D Systems, Abingdon, UK; AF980, AF971, AF8520, and MAB901, respectively) were utilized.

Techniques: Expressing, Western Blot, Standard Deviation, Control