Live-cell ERK Activity Measurement

We used SH-SY5Y:ERK-KTR cell line established as described in [39 (link)] to measure ERK activity in live neuroblastoma cells. Briefly, ERK-KTR translocation reporter was introduced into SH-SY5Y cells through lentiviral transduction. For that purpose, we used pLentiCMV Puro DEST ERKKTRClover plasmid (Addgene plasmid #59150), which encoded ERK docking domain ELK, nuclei localization site and nuclei extraction site (containing phosphorylation sites) fused with fluorescent protein mClover [40 (link)]. Lentiviral particles were collected with the culture medium of HEK-293T cells 24 and 48 h after their transfection with plasmids coding structural elements of the lentivirus as described at Lentiviral Gene Ontology Vectors [41 ]), plasmid coding G protein (envelope) of the vesical stomatitis virus VSV and #59150 plasmid. Cells were imaged with Leica DMI8 automated microscope using 10× magnification lenses. Nuclei of the cells were stained with 500 ng/mL Hoechst-33342 for 30 min before imaging. For each drug/combination of drugs we imaged three wells and three fields in each well. Imaging of the cells was performed in two channels-461 nM (for Hoechst) and 520 nM (for mClover). Illumination correction, segmentation of nuclei and cells, calculation of cytoplasm to nucleus ratios of individual cells (C/N ratio) corresponding to ERK activity were made in CellProfiler.