Puromycin

pGF1-GAS-LUC, which expresses a puromycin selection cassette and firefly luciferase reporter under the control of a minimal CMV promoter followed by four tandem consensus GAS elements (5′-AGTTTTCATATTACTCTAAATC-3′) was purchased from System Biosciences (SBI). Reporter vector carrying viral particles was produced by co-transfection of the lentiviral plasmid and the packaging vectors into Lenti-X 293T cell line (Clontech). Ulk1/2^+/+ and Ulk1/2^−/− MEFs were infected with virus-containing fresh supernatant using Transdux reagent (SBI). Stably-transduced cells were selected using 2 μg/ml of puromycin (Gibco, Life Technologies).

4T1 cells were procured from the American Type Culture Collection (ATCC) and grown in 75 cm² culture flasks to 80% confluence in Dulbecco’s Modified Eagle Media with high glucose and glutamine (Thermo Fisher). Cells were then dispersed by removing media, washing briefly with Dulbecco’s PBS and treating with trypsin for 15 min. A modified version of the PBQM812A-1 vector (System Biosciences) was used to insert a cumate inducible expression cassette, along with a puromycin resistance selection marker, into the 4T1 genome using the piggyBAC transposon system. This vector was modified to induce expression of a His-tagged human EGFR fused via to a T2A self-cleaving linker to GFP at its C-terminus. This vector was first complexed with lipofectamine (Thermo Fisher) before being added to the 4T1 cells in suspension. These transfected cells were then plated on 75 cm² flasks and selected for with puromycin (puromycin Dihydrochloride, Thermo Fisher) starting at 1 μg/mL for several passages and ending with 2 μg/mL. Transfected cells were additionally induced via 15 μg/mL cumate (Sigma-Aldrich) added to the culture media and then sorted for GFP expression via FACS. A subpopulation consisting of the top 5% most fluorescent cells were used in all future experiments.