Growth factor-induced ERK activity pulsing dynamics in a mammary epithelial cell line. Dataset 3 of 3: mean reporter pulsing metrics. - Dataset (ID:20302)
|HMS Dataset ID:||20302|
|Dataset Title:||Growth factor-induced ERK activity pulsing dynamics in a mammary epithelial cell line. Dataset 3 of 3: mean reporter pulsing metrics.|
|Screening Lab Investigator:||Somponnat Sampattavanich, Bernhard Steiert|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||ERK activity as measured by the EKAREV fluorescent reporter was 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.|
1. Cells stably expressing EKAREV and H2B-mCherry reporters were plated in 96-well plates at ~6 x 105 cells/cm2 and cultured at 37°C with 5% CO2 in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, 20 ng/mL EGF, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, 50 U/mL penicillin, and 50 µg/mL streptomycin (PMID: 12372298).|
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.
3. The cells were imaged using a 10x objective on a Nikon Eclipse inverted fluorescence microscope fitted with an environmental chamber maintained at 37°C with 5% CO2. Images were collected at 5 min intervals using a Hamamatsu ORCA-ER cooled CCD camera and Spectra-X light engine (Lumencor).
4. At 67.4 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.
5. Cell tracking was performed using a custom MATLAB script for cross-correlation between adjacent frames with manual validation. Image segmentation was performed using H2B-mCherry nuclear reporter signal.
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 #20300).
7. The fPCA harmonics from HMS LINCS Dataset #20287 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.
8. To calculate pulsatile score, preprocessing was utilized to bowdlerize artifacts from traces such as spikes resulting from e.g. cell division or loss of tracking. A sliding window subtraction of 10 data points length was applied. Missing values were added by interpolation. The first three fPCA harmonics were employed to remove the slow time scales and thus detrend the signal. Then, the detrended signal was smoothed using N/3 b-splines for N data points. 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 fi:
(1) number of edges (approximately 2x the number of peaks)
(2) range (min-to-max)
(3) signal-to-noise ratio = range/(1/(N-1)*sqrt(RSS)) with RSS = distance of spline to data
(4) peak duration = mean(edge_start to neighboring_edge_end)
(5) peak distance = mean(min to neighboring min; max to neighboring max)
Whenever no peaks were detected, peak duration and peak distance were set to 300 min. A reference value ri and a weight wi were defined for all features fi. A positive value for wi was chosen for features where a larger number represents more pulsing, i.e. nEdges, range, and signal-to-noise ratio. A negative number for wi was assigned to features where a larger number indicates less pulsing, i.e. peak duration and distance. Using the features fi, the reference values ri, and the weights wi, pulsatile score
was calculated and compared to a threshold for assigning each trace into either the pulsing or the non-pulsing group (HMS LINCS Dataset #20301).
9. 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
was calculated (HMS LINCS Dataset #20302).
10. The complete set of images for this dataset is viewable online through our OMERO server at omero.hms.harvard.edu/webclient/?show=plate-907, and a lookup table with data file data column definitions is contained within the downloadable data package for this dataset.
|HMS Dataset Type:||Analysis|
|Date Publicly Available:||2017-05-02|
|Most Recent Update:||2017-05-02|