Fugene 6 reagent

siRNAs (Supplementary Table S2) and plasmid DNA was transfected into EpiSCs using the Fugene6 reagent (Promega). Equal numbers of cells were seeded on 6-well plates and transfected within 4 h with the different siRNAs (100 pmol per well) or plasmid DNA (4 μg per well). siRNAs, plasmids and transfection reagent were diluted in CDM. Cells were harvested 36 h or 96 h post-transfection. Cells were stably transfected with the Cre recombinase expression vector pCAGGS-CreERT236 (link). Induction of recombination was carried out by adding 0.1 μg/ml Tamoxifen to the medium for three days.

The recruitment of β-arrestin2 was measured using the PathHunter β-arrestin assay (DiscoverX) as described previously (Daugvilaite et al., 2017 (link)). Briefy, cDNA encoding US28wt was fused to the ProLink C-terminal protein tag and the small fragment ofβ-galactose and cloned into pcDNA3.1+. Assays were performed using the CHO-K1 EA-arrestin cell line with the stable expression of β-galactosidase coupled to the β-gal large fragment. Cells were seeded at 2 × 10⁴ cells per well and transfected the next day with 50 ng DNA using Fugene6 reagent (0.15 µl per well, Promega, Madison WI). 48 hr post transfection cells were stimulated with various concentrations of the chemokine for 90 min (as positive control CCR5 transient transfected cells were included, stimulated with CCL5). β-arrestin2 recruitment was detected as β-gal activity using the PathHunter detection kit (DiscoverX). Chemiluminescent substrate composed of Galacton Star Substrate, Emerald II Solution and PathHunter Cell Assay Buffer in a ratio of 1:5:19, respectively, was added to the cells (50 µL per well). The luminescent signal was determined after 60 min incubation at ambient temperature using the EnVision Multilabel Plate Reader (PerkinElmer, Waltham MA).