Mycoplasma contamination

All cells were cultured in a 5% CO₂ atmosphere at 37°C. MRC-5 (ECACC 05090501), HFF (HF-99/7 kindly provided by Dieter Neumann-Haefelin and Valeria Kapper-Falcone, Institute of Virology, Freiburg Germany), 293 T-CD20 (kindly provided by Irvin Chen, UCLA USA [Morizono et al., 2010 (link)]), BJ-Her2 (BJ-5ta foreskin fibroblasts [ATCC CRL-4001] stably expressing Her2/Erbb2 (NM_004448), lentiviral transduction as in Halenius et al., 2011 (link)), and Hela cells (ATCC CCL-2) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (vol/vol) fetal calf serum (FCS, Biochrom). BW5147 mouse thymoma cells (kindly provided by Ofer Mandelboim, Hadassah Hospital, Jerusalem, Israel) were maintained at 3 × 10⁵ to 9 × 10⁵ cells/ml in Roswell Park Memorial Institute medium (RPMI GlutaMAX, Gibco) supplemented with 10% (vol/vol) FCS, sodium pyruvate (1×, Gibco) and β-mercaptoethanol (0.1 mM, Sigma). Cells were used when tested negative for mycoplasma contamination (Eurofins). If positive, cells were treated using Plasmocure (Invivogen) or BM-Cyclin (Roche) as instructed by the supplier.

No cell lines used in this study were found in the database of commonly misidentified cell lines (maintained by ICLAC and NCBI Biosample). Ins1E cells were regularly tested negative for mycoplasma contamination (Eurofins) and their identity confirmed by functional insulin secretion assays, qPCR, immunofluorescence stainings and western blots for β-cell marker mRNAs/proteins. Ins1E cells (including knockout cell lines) were cultivated in RPMI 1640 with 10% FCS, 2 mM Glutamax, 10 mM HEPES, 2 mM Na-pyruvate, 0.18 mM 2-mercaptoethanol and 100 U ml⁻¹ penicillin–streptomycin (all Thermo Fisher). CRISPR knockout lines were engineered according to a double single guide RNA (sgRNA) strategy²⁹ using the PX459 plasmid or a modified PX458 plasmid^{55 (link)}. pSpCas9(BB)−2A-GFP (PX458) and pSpCas9(BB)−2A-Puro (PX459) were a gift from Feng Zhang (Addgene plasmids #48138 and #48139). Positive clones were enriched using either puromycin selection or FACS sorting using a FACSAria III (BD Biosciences, running FACSDiva software) or a Cytoflex SRT (Beckman Coulter, running CytExpert software). Monoclonal colonies were grown until near confluency in a 96-well plate, and after passaging, knockout was confirmed using deletion PCR, qPCR and/or immunoblot. At least three monoclonal cell lines were pooled before experiments^{56 (link)}, if not denoted otherwise in the figure legends.