Lentiviral and retroviral transduction

Lentiviral particles were produced using HEK293T cells and a third-generation packaging system, MISSION® Lentiviral Packaging Mix, per the manufacturer’s recommendations (Sigma Aldrich). To stably express specific shRNAs, MEF, or U2OS cells were transduced with optimized titers of lentiviruses. Next day medium was changed, and cells were allowed to recover for 24 h before antibiotic selection (2 µg/mL of puromycin) for 3–6 days. A lentiviral CRISPR-Cas9 [32–34] construct containing sgRNA against βarr1 was used to generate U2OS βarr1 KO cell lines, mRNA expression measured by real-time PCR and western blot analysis confirmed deletion of βarr1 expression in the selected clones.

Retroviral transduction was performed with ecotropic viruses. In brief, for the production of virus HEK293T cells were transiently transfected with pcl ECO vector (which expresses the viral genes gag, pol, and envEco) and the vector of interest (1:1 ratio). After 48 h, the supernatant containing the viral particles was diluted 1:2 with fresh medium, filtered, and polybrene was added to a final concentration of 5 µg/mL; MEF cells were transduced with this mix. This procedure was repeated 3 times, every 4 h. Next day medium was changed, and cells allowed to recover for 24 h before antibiotic selection (2 µg/mL of puromycin) for 3–6 days.