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Dataset Information
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Dataset Name:
Growth factor-induced ERK activity and FoxO3a nuclear-cytoplasmic pulsing dynamics in a mammary epithelial cell line. Dataset 1 of 1: single-cell reporter measurements.

Dataset Description:
ERK activity and the nuclear-cytoplasmic pulsing behavior of FoxO3a as measured by a dual fluorescent reporter (EKAREV-FoxO3aN400-mCherry) were assessed in the MCF 10A mammary epithelial cell line at the single-cell level using live imaging of cells that were untreated or treated with one of six growth factors.

--Data in Package:
20299.txt

--Metadata in Package:
Protein_Metadata.txt
Cell_Line_Metadata.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20299

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20299/

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Assay Information
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Assay Protocol:
1. Cells stably expressing a EKAREV-P2A-FoxO3aN400-HA-mCherry dual reporter (in which P2A acts as a self-cleaving peptide between the two reporters) were plated in 96-well plates at ~6 x 10<sup>5</sup> cells/cm<sup>2</sup> and cultured at 37C with 5% CO<sub>2</sub> in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, 20 ng/mL EGF, 10 g/mL insulin, 0.5 g/mL hydrocortisone, 100 ng/mL cholera toxin, 50 U/mL penicillin, and 50 g/mL streptomycin (PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/12372298" target = "_blank">12372298</a>).<br />
2. Prior to growth factor addition, the cells were washed twice with PBS, placed in serum-free medium (DMEM/F12 with penicillin/streptomycin and no phenol red) for 6 hr, washed again, and placed in fresh serum-free medium.<br />
3. The cells were imaged using a 10x objective on a Nikon Eclipse inverted fluorescence microscope fitted with an environmental chamber maintained at 37C with 5% CO<sub>2</sub>. Images were collected at 5 min intervals using a Hamamatsu ORCA-ER cooled CCD camera and Spectra-X light engine (Lumencor).<br />
4. At 48 min, imaging was briefly paused. Growth factor diluted in serum-free medium was added within 1 minute, resulting in an ~5% increase in culture volume, and imaging was resumed and continued for 24 hr.<br />
5. Cell tracking was performed using a custom MATLAB script for cross-correlation between adjacent frames with manual validation. Image segmentation was performed based on the YFP channel using edge-based thresholding.<br />
6. To quantify ERK activity, background signal was subtracted, and the FRET/CFP ratio from the EKAREV reporter was calculated at the single pixel level. Single-cell activity is reported as the mean FRET/CFP ratio per pixel in the nuclear region and the mean FRET/CFP ratio per pixel in the cytosolic region dilated by 4 pixels from the nuclear mask (HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20299/">#20299</a>).<br />
7. Quantification of FoxO3a level in each cell is reported as the mean fluorescent intensity per pixel in the cytosolic region, the mean in the nuclear region, and the log<sub>10</sub> ratio of mean cytosolic to nuclear fluorescent intensity (HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20299/">#20299</a>).<br />
8. To calculate normalized signals for comparison, cytosolic EKAREV signal was scaled to range between [0,1] and detrended by fPCA, and the log<sub>10</sub> of the ratio between cytoplasmic and nuclear fluorescent intensity for the FoxO3a reporter was scaled to range [0,1], outliers removed, and detrended by fPCA (HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20299/">#20299</a>).<br />
9. The complete set of images for this dataset is viewable online through our OMERO server at <a href="https://omero.hms.harvard.edu/webclient/?show=plate-909"target = "_blank">omero.hms.harvard.edu/webclient/?show=plate-909</a>, and a lookup table with data file data column definitions is contained within the downloadable data package for this dataset.

Date Updated:
2017-05-02

Date Retrieved from Center:
11/13/2015

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
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