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Dataset Information
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Dataset Name:
IGF-I-induced FoxO3a nuclear-cytoplasmic pulsing dynamics in mammary epithelial cell lines following treatment with increasing doses of an AKT inhibitor. Dataset 3 of 3: mean reporter pulsing metrics.

Dataset Description:
The nuclear-cytoplasmic pulsing behavior of a FoxO3a fluorescent reporter (FoxO3aN400-HA-Venus) expressed in two mammary epithelial cell lines was assessed at the single-cell level using live imaging of cells that were untreated or treated with IGF-I in the absence or presence of increasing doses of an AKT inhibitor.

--Data in Package:
20295.txt

--Metadata in Package:
Small_Molecule_Metadata.txt
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:
20295

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20295/

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Assay Information
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Assay Protocol:
1. Cells stably expressing a FoxO3aN400-HA-Venus-P2A-NLS-Myc-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 drug and 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 5 hr, washed again, and placed in fresh serum-free medium.<br />
3. DMSO or the AKT inhibitor MK2206 was added.<br />
4. 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 />
5. At 60 min after drug addition, imaging was briefly paused. IGF-I 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 />
6. Cell tracking was performed using a custom MATLAB script for cross-correlation between adjacent frames with manual validation. Image segmentation was performed using NLS-Myc-mCherry nuclear reporter signal.<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/20293/">#20293</a>).<br />
8.  The fPCA harmonics from HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20287/">#20287</a> were used to determine fPC scores for the time interval t = [-70,80] min. These fPC scores represent the projections of the measured data onto the fPCA harmonics.<br />
9. To calculate pulsatile score for times t > 80 min, preprocessing was utilized to bowdlerize artifacts from traces such as spikes resulting from e.g. cell division or loss of tracking. Missing values were added by interpolation. The first three fPCA harmonics of the late response from HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20287/">#20287</a> were employed to remove the large time scales. Peaks were detected on the smooth data representation. Due to overfitting, the pulsatile traces are mixed with small artificial peaks. These were dropped if the peaks were smaller than a certain threshold. The final detrended, interpolated, and peak-adjusted signals were then lumped into the following features <i>f<sub>i</sub></i>:<br />
(1) number of edges (approximately 2x the number of peaks)<br />
(2) range (min-to-max)<br />
(3) signal-to-noise ratio = range/(1/(N-1)*sqrt(RSS)) with RSS = distance of spline to data<br />
(4) peak duration = mean(edge_start to neighboring_edge_end)<br />
(5) peak distance = mean(min to neighboring min; max to neighboring max)<br /><br />
Whenever no peaks were detected, peak duration and peak distance were set to 300 min. A reference value <i>r<sub>i</sub></i> and a weight <i>w<sub>i</sub></i> were defined for all features <i>f<sub>i</sub></i>. A positive value for <i>w<sub>i</sub></i> was chosen for features where a larger number represents more pulsing, i.e. nEdges, range, and signal-to-noise ratio. A negative number for <i>w<sub>i</sub></i> was assigned to features where a larger number indicates less pulsing, i.e. peak duration and distance. Using the features <i>f<sub>i</sub></i>, the reference values <i>r<sub>i</sub></i>, and the weights <i>w<sub>i</sub></i>, pulsatile score<br /><br />
<center><img src="/_static/dataset_images/20290-92_equation-1.png"/></center><br />
was calculated and compared to a threshold for assigning each trace into either the pulsing or the non-pulsing group (HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20294/">#20294</a>).<br />
10. Finally, mean fPCA values and features were calculated across all single-cell traces for a given condition, and the fraction of pulsing cells per condition<br />
<center><img src="/_static/dataset_images/20290-92_equation-2.png"/></center><br />
was calculated (HMS LINCS Dataset <a href="http://lincs.hms.harvard.edu/db/datasets/20295/">#20295</a>).<br />
11. 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-803"target = "_blank">omero.hms.harvard.edu/webclient/?show=plate-803</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/
[/generic/reagents_studied]
