Journal: Oncogene

Article Title: α3β1 integrins regulate CD151 complex assembly and membrane dynamics in carcinoma cells within 3D environments.

doi: 10.1038/onc.2012.415

Figure Lengend Snippet: Figure 6. a3 integrin promotes protrusion in cells in 3D ECM. (a) Representative images of cells expressing control or a3 integrin shRNA were co-transfected with Lifeact–GFP (LA–GFP) and CD151–RFP and embedded in 3D native collagen I (COL I) or matrigel (MG) within imaging chambers. Cells were left to invade for 24 h and were then fixed and subjected to confocal microscopy. (b) Representative confocal z-stack projections of control cells expressing Lifeact–GFP and embedded within cy3-labelled COL or MG 3D gels. (c) Representative 3D projections of confocal z-stacks of control or a3 integrin shRNA cells expressing Lifeact–GFP and invading into 3D COL or MG gels. Images are stills taken from movies shown in Supplementary data. (d) Analysis of dynamics of Lifeact–GFP protrusions from cells as shown in example images in c. Protrusion number per cell was calculated for each time point over the duration of the movie (d). n ¼ 8 cells per condition over three independent experiments. (e) Graph showing change in protrusion volume as a % of total cell volume over time from analysis of movies as in (d). (f) Protrusion: retraction ratio as calculated from protrusion dynamics data from live cell analysis as in d and e. *P ¼ 0.01, **P ¼ 0.005 compared with shCon. Scale bars, 10 mm.

Article Snippet: 1 ml of diluted collagen or laminin was added to an aliquot of Cy3 label from a Cy3 Mono-Reactive Dye Pack (GE Healthcare, Bucks, UK) and mix/rotated for 30 min at 41C in the dark.

Techniques: Expressing, Control, shRNA, Transfection, Imaging, Confocal Microscopy, Cell Analysis

Journal: Journal of Biological Chemistry

Article Title: Proteomic Analysis of DC-SIGN on Dendritic Cells Detects Tetramers Required for Ligand Binding but No Association with CD4

doi: 10.1074/jbc.m402741200

Figure Lengend Snippet: FIG. 6. FRET measurements between CD4 and DC-SIGN on iMDDCs and between CD4 and CD71 on CemT4 cells and iMDDCs. Energy transfer efficiencies were measured using different fluorescein conjugated antibodies against CD4 (OKT4 and Q4120) as donor antibodies and Cy3-conjugated antibodies against DC-SIGN, CD4 (OKT4) and the TFR (WM82). Energy transfer efficiencies were calculated as the percentage of donor fluorescence quenching in the presence of an acceptor antibody and were measured either before (mock) stimulation (dark bars) and after 15 min incubation with or without a stimulus (shaded bars). Shown are the mean energy transfer efficiencies of ten independent experiments (OKT4/DC-SIGN) or four independent experiments (all others). A significant energy transfer (5% to 7%) can only be detected on CemT4 cells between CD4 and the TFR after PMA stimulation. No significant transfer was detected for this interaction on iMDDCs and between CD4 and DC-SIGN on iMDDCs.

Article Snippet: Disuccinimidyl suberate (DSS), dithiobis(succinimidylpropionate) (DSP), were from Pierce, protein G-Sepharose and Cy3 monoreactive NHS ester from Amersham Biosciences.

Techniques: Fluorescence, Incubation