Code and Data, Ram et al.
We generated a dataset that enables correlation of ERK activation to downstream target protein expression and modification by first collecting live ERK activity measurements under differential activation states of the RAS/ERK pathway. In MCF10A mammary epithelial cells, MCF7 breast cancer cells, and Hcc827 and A549 lung adenocarcinoma cells, we used EKAR3.1, a calibrated FRET-based biosensor that measures the balance between phosphorylation by ERK activity and dephosphorylation by competing phosphatases. ERK activity was stimulated in a dose-dependent manner with varying Epidermal Growth Factor (EGF) concentrations. To increase the temporal diversity of activity patterns, we added MEK inhibitor (MEKi) at varying times after EGF stimulation and included treatments where EGF was added at different timepoints of the experiment. These treatments generated a wide range of ERK signaling patterns, including sustained and pulsatile activity with varying duration and magnitude. Consistent with previous studies, we found that ERK activation is heterogenous from cell to cell within each stimulation condition. Immediately following live-cell data collection, we fixed the cells and conducted cyclic immunofluorescence (4i) staining to measure nuclear and cytoplasmic levels of protein targets potentially modulated by ERK activity.
This repository contains all code and data needed to generate the figures in the paper.
Quick start guide (see the README file for more detailed instructions):
-Typical install time is about 30 minutes to one hour.
-Unzip Code_files.zip to view the custom code associated with processing and plotting data.
-To access the source MCF10A supplementary data tables, unzip SourceData.zip (in the same folder as you unzipped Code_files.zip)
-To access the (large) cancer cell line data files needed to run the figure generation code, unzip Data_files.zip WITHIN the MATLABfiles subfolder that was extracted from Code_files.zip
Funding
Decoding temporal epithelial signaling programs to restore homeostasis in acute lung injury
National Heart Lung and Blood Institute
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National Institute of General Medical Sciences
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