Journal: World Journal of Stem Cells

Article Title: Circulating factors present in the sera of naturally skinny people may influence cell commitment and adipocyte differentiation of mesenchymal stromal cells

doi: 10.4252/wjsc.v11.i3.180

Figure Lengend Snippet: Cytokines detection in sera. The histogram shows the cytokines on the Human Obesity Antibody Array C1 (RayBiotech, Norcross, GA United States) that were detected in SP and NP sera. Array signals were acquired using the Chemidoc system (Bio-Rad) and the associated software QuantityOne. Data are expressed as arbitrary units ( n = 3 ± SD, a P < 0.05, b P < 0.01). The picture shows only the cytokines whose expression was above the limit of quantification. NP: Normal people; SP: Skinny people; SAA: Serum amyloid A; RANTES: CCL5; IGFBP-1: Insulin-like growth-factor-binding protein-1.

Article Snippet: We acquired array signals with the Chemidoc system (Bio-Rad Company, United States) and the related software QuantityOne.

Techniques: Ab Array, Software, Expressing, Binding Assay

Journal: Frontiers in pharmacology

Article Title: Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion.

doi: 10.3389/fphar.2015.00202

Figure Lengend Snippet: FIGURE 5 | Characterization of cAMP-PDEs in human CTs. (A) CTs purified from human placenta were transfected with a DNA construct directing the expression of the TEpacVV biosensor and imaged with wide field microscopy. Images show the fluorescence at 535 nm (upper left, in gray scale) and the ratio (in pseudocolor; lower row) indicating a ratiometric change of TEpacVV, reporting the binding of cAMP at the times indicated by arrows on the graph (upper right panel). The trace on the graph indicates the F480/F535 emission ratio measurement on region indicated in red on the gray scale image. Human CG (1 μM) induced a robust ratio increase in CTs. FSK + IBMX incubation were used to induced the maximal response. Data are representative of three separate experiments (B) cAMP-PDE mRNA expression profile in CTs. Total RNA was purified from CTs (n = 5 cultures) and subjected to RT-qPCR, using Human PDE TaqMan Gene Expression Assays chip (Applied Biosystems). Histograms represent mRNA expression of cAMP-PDEs in trophoblast normalized to 18S rRNA expression. (C) Cyclic AMP-PDE activity in human CTs. LANCE Ultra cAMP assay (Perkin Elmer) was adapted to quantify PDE activity in total CT lysates. Trophoblast cell lysates (20 μg) were incubated in presence of 6 nM cAMP with or without PDE antagonists, subsequently (Eu)-cAMP tracer and ULight-labeled anti-cAMP antibody were added to the solution prior to analysis. Histograms represented the profile of PDE activity in trophoblasts. (D) Effect of PDE inhibitor on CT fusion. Cytrophoblasts were stimulated with hCG (1 μM) and treated with pan-PDE (IBMX, 200 μM), PDE4 (Rolipram, 4 μM) or PDE3 (Cilostamide, 100 nM) antagonists. Cells were stained for desmoplakin (magenta) and nuclei (DAPI, left), Syncytia (ST) boundaries are indicated by dashed lines and fusion indices were measured (right histograms). Results are expressed as the mean ± SEM of indicated independent experiments (n.s., non significant, ∗p < 0.05, ∗∗∗p < 0.001). Scale bar: 30 μm.

Article Snippet: Reverse transcription-polymerase chain reaction (RT-PCR) was performed with the TaqMan R©Array Human Phosphodiesterase (4414075, Applied Biosystem) and Human PrimePCR Phosphatases (H384, Biorad) or primers ordered from SantaCruz Biotechnology according to the manufacturers’ protocols for analysis of PDEs, PPs, and AKAPs, respectively.

Techniques: Transfection, Construct, Expressing, Microscopy, Binding Assay, Incubation, Quantitative RT-PCR, Gene Expression, Activity Assay, cAMP Assay, Labeling, Staining