Opti mem 1 1 reduced serum medium

MEFs were plated at 1 × 10⁶ cells per 10-cm dish in DMEM with 10% FBS. The next day, indicated cell lines were transfected with 1.5 µg of either insertless vector pcDNA 3.1+ (Invitrogen) or human HRAS (V12) expression plasmid (a gift from Dr. Michael Cole, Geisel School of Medicine at Dartmouth) or wild-type EGFR expression plasmid (Addgene 11011) along with 1.0 µg of GFP expression plasmid pEGFP-C1 (Clontech) to control for transfection efficiency. Transfections were performed with EndoFectin™-Lenti (GeneCopoeia) and Opti-MEM I (1×) Reduced Serum Medium (Life Technologies) according to the manufacturers’ protocols. The day after transfection, cells were split into 10-cm dishes and GFP expression was determined with a florescent microscope the following day. Cells were allowed to grow to confluence and media was changed every 3 days. Cells were fixed with methanol and stained with Giemsa 14 days after transfection to visualize transformed foci. The number of foci was manually counted. For G0S2 co-transfection foci assay, MEFs were transfected with 1.5 µg of HRAS (V12) expression plasmid and either 7.0 µg of insertless vector pcDNA 3.1+ or human G0S2 expression plasmid pCMV-SPORT6-G0S2 (Thermo Fisher). For co-transfection of shRNA, 1.5 µg HRAS (V12) was co-transfected with 5.0 µg of shMYC, shATGL or corresponding shControl lentiviral expression plasmid.