CRISPR/Cas9 Knockout and WT HeLa Cell Lines

Stable CRISPR/Cas9 μ1A KO and WT HeLa cells were a gift from Margaret S. Robinson (University of Cambridge) and have been previously described (20 (link)). H4 (human neuroglioma) cells were obtained from the American Type Culture Collection. PEAK cells are HEK-293T cells transfected with the large T antigen of SV-40 (59 ) and were used for co-immunoprecipitation experiments. To generate HeLa cell lines expressing ER-targeted streptavidin fused to KDEL (ER hook), retrovirus coding streptavidin-KDEL was produced in HEK-293T cells and used to transduce HeLa cells. Transduced cells were selected by incubation in complete media in the presence of 1 μg/ml puromycin. Cells were maintained as previously described (60 ). DNA transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific). The siRNAs were purchased from Dharmacon as nucleotide duplexes with 3′dTdT overhangs, designed to target human γ1 (5′-GGAAGAGCCUAUUCAGGUA-3′) (51 (link)). Transfections of siRNA were performed in two rounds with an interval of 48 h between treatments using Oligofectamine reagent (Thermo Fisher Scientific).