KT-1 cells were transduced with Edit-R Lentiviral hEF1α-Blast-Cas9 Nuclease Plasmid DNA and Edit-R Human ULK1 Lentiviral sgRNA (no. CAS10138 and no. VSGH10142-246477203, GE Healthcare Dharmacon). Transduction was carried out by spinoculation (750g) for 90 min at room temperature in the presence of Transdux reagent (SBI), followed by 48 hours of incubation. Stably transduced cells were selected using blasticidin (5 μg/ml) and puromycin (2.5 μg/ml) antibiotics. One week later, antibiotic-resistant cells were separated from dead cells using a BD FACSAria 5-Laser Cell Sorter (BD Biosciences) and performed at the Northwestern University Flow Cytometry Core Facility. Lack of ULK1 protein in KT-1 ULK1 KO cells was confirmed using Western blot analysis.

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