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Results: 7

1.
Fig. 4

Fig. 4. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

DQ-collagen IV is degraded as HUVECs migrate in vitro into cord-like structures. HUVEC were grown for 18 h on glass coverslips coated with rBM containing 25 μg/ml DQ-collagen IV. Panel a illustrates a DIC image of the HUVEC cord-like structures at 18 h after plating. Panels b, c and d are confocal images of live HUVECs at 2, 6 and 14 h, respectively, after plating. DQ-collagen IV degradation products (green) are seen pericellularly adjacent to the cells. Bars, 100 μm

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
2.
Fig. 5

Fig. 5. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

Isogenic MCF-10A variants model premalignant progression when grown in 3D rBM overlay cultures. a Polarized 10A acinus; bar, 10 μm. b Stages of normal, atypical hyperplasia, dysplasia and carcinoma; bar, 50 μm. At 12 days, the structures were fixed and processed. Images represent a single confocal section at the equatorial plane. Structures were stained for: α6 integrin, as a marker of basal polarity (red); DAPI, as a marker of nuclei (blue); and cleaved caspase-3, as a marker of apoptosis (green)

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
3.
Fig. 6

Fig. 6. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

2D imaging in real-time of breast fibroblasts degrading DQ-collagen I (green fluorescence) as they migrate on a matrix of collagen I containing DQ-collagen I. Note trail of fluorescent cleavage products (denoted in panel b and the inset by arrows) left behind by migrating fibroblast. Round cells represent cells that are in process of detaching prior to migrating. Confocal images were taken over a 90 min period starting 2 h after plating (panel a, 0 min; panel b, 90 min). Bar, 10 μm

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
4.
Fig. 3

Fig. 3. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

Inhibitors of MMPs (GM6001), cysteine cathepsins (CA074/CA0Me) and serine proteases (aprotinin) reduce degradation of DQcollagen IV (green fluorescence) in co-cultures of BT549 breast carcinoma cells and WS-12Ti fibroblasts to levels below that in cultures of the tumor cells alone. Images illustrate top views of cultures from which optical sections were acquired. Fluorescent intensity per cell, represented as bar graphs above each image, was quantified with Metamorph 6.3 software (Molecular Devices) in Zstacks of optical sections; nuclei were stained with Hoechst (blue fluorescence) at the time of imaging and counted in Z-stacks reconstructed in 3D, using Volocity 3.6.1 software (Improvision) [62]. WS-12Ti fibroblasts were prelabeled with Cell Tracker Far Red (Invitrogen). Bar, 10 μm

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
5.
Fig. 1

Fig. 1. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

Diagrammatic representation of the components of a 3D rBM ‘overlay culture’ grown on rBM containing a DQ-substrate (a) and of the tripartite MAME model (b). Green, red and dark blue represent the fluorescent cleavage fragments of the DQ-substrate, cells labeled with a cytoplasmic dye and nuclei stained with a DNA binding dye, respectively. The invasion of cells into the underlying ECM and pericellular and intracellular (green spots adjacent to nuclei) proteolysis are depicted. Imaging in our laboratory is primarily done on an upright microscope using a dipping lens as represented here. The tripartite MAME model (b) includes a lower stromal layer, which consists of fluorescently pre-labeled fibroblasts embedded in a collagen I matrix containing DQ-collagen I. Note the use of a plastic coverslip in order to promote adherence of the collagen I matrix. A 3D rBM overlay culture of breast cells (e.g., isogenic 10A variants) is layered on top of the stromal layer

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
6.
Fig. 2

Fig. 2. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

Tumor cell degradation of ECM is increased when the tumor cells are co-cultured with fibroblasts. Images illustrate degradation fragments of DQ-collagen IV (green fluorescence) in live 3D cultures of HCT 116 human colon carcinoma cells (a1–3), BT549 human breast carcinoma cells (b1–3) and BT20 human breast carcinoma cells (c1–3). Pericellular degradation of DQ-collagen IV is increased when HCT 116 cells are co-cultured with CCD-112CON normal colon fibroblasts (a3) and BT549 or BT20 cells are cocultured with WS-12Ti breast carcinoma-associated fibroblasts (b3 and c3, respectively). (Collagen IV degradation by monocultures of the two fibroblast lines can be observed in Fig. 5 of [14].) Tumor cells were seeded alone or with fibroblasts that had been prelabeled with Cell Tracker Orange (Invitrogen). Starting at 1 h after seeding, live cells were imaged by confocal microscopy over a total time period of 24 h and representative confocal sections at the equatorial plane are shown at 2 h (a1, b1 and c1) and 24 h (a2, b2, c2, a3, b3 and c3). Bar, 20 μm

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.
7.
Fig. 7

Fig. 7. From: Imaging and quantifying the dynamics of tumor-associated proteolysis.

Quantification of DQ-collagen IV degradation products, including extracellular and intracellular products, generated by live HCT 116 human colon carcinoma cells in 3D rBM culture. Total proteolysis of DQ-collagen IV in all optical sections throughout the volume of the structure was determined as normalized integrated intensity based on cell number. Total fluorescence was separated into pericellular and intracellular degradation using image arithmetic in Metamorph 6.3 software. Optical sections were reconstructed in 3D Z-stacks of confocal sections with Volocity 3.6.1 software. a Single optical section taken at equatorial plane showing fluorescence from cleaved DQ-collagen IV (green), cells pre-labeled with Cell Tracker orange (pseudocolored red) and nuclei stained with Hoechst (blue) at the time of imaging. b Cleavage products of DQ-collagen IV from same optical plane, representing both intracellular and pericellular cleavage products of DQ-collagen IV. c Pericellular cleavage products of DQ-collagen IV obtained by masking image from panel b with a binarized image of the cells used to designate x, y coordinates of intracellular areas within the same optical section. This eliminates all of the signal from cytoplasmic areas. d Image of intracellular cleavage products of DQ-collagen IV obtained by subtracting the image in panel c from that in panel b. A binarized image of nuclei was used to create a 3D reconstruction for counting nuclei, thus allowing us to determine the number of cells within the field of view. Bar, 10 μm

Mansoureh Sameni, et al. Clin Exp Metastasis. ;26(4):299-309.

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