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 (ID:20299)
| HMS Dataset ID: | 20299 |
| Dataset Title: | 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. |
| Screening Lab Investigator: | Somponnat Sampattavanich, Bernhard Steiert |
| Screening Principal Investigator: | Peter K. Sorger |
| Assay 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. |
| 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 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 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. 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. 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 #20299). 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 log10 ratio of mean cytosolic to nuclear fluorescent intensity (HMS LINCS Dataset #20299). 8. To calculate normalized signals for comparison, cytosolic EKAREV signal was scaled to range between [0,1] and detrended by fPCA, and the log10 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 #20299). 9. The complete set of images for this dataset is viewable online through our OMERO server at omero.hms.harvard.edu/webclient/?show=plate-909, and a lookup table with data file data column definitions is contained within the downloadable data package for this dataset. |
| HMS Dataset Type: | Microscopy/Imaging |
| Date Publicly Available: | 2017-05-02 |
| Most Recent Update: | 2017-05-02 |