Plenticmv puro dest erkktrclover plasmid

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.

An amount of 10⁶ HEK-293T cells were plated on 100 cm² Petri dishes 24 h before the transfection. Calcium transfection was used to deliver plasmids into the HEK-293T cells. Cell culture media containing lentiviral particles was collected from HEK-293T cells 24 and 48 h after the transfection with plasmids, coding structural elements of the lentivirus (as described at http://www.lentigo-vectors.de, accessed on 16 October 2022), plasmid coding G protein (envelope) of the vesical stomatitis virus VSV, and a vector plasmid containing a gene of interest. To obtain ERK-KTR cell lines for ERK quantification, pLentiCMV Puro DEST ERKKTRClover plasmid (Addgene, #59150) was used, which encoded the ERK docking domain ELK, nuclei localization site, and nuclei extraction site (with phosphorylation sites) fused to fluorescent protein mClover.