Cell Culture and Genetic Manipulation Protocols

Human BT-549 TNBC, A549 (mutant KRAS) NSCLC, H460 (mutant KRAS) NSCLC and embryonic kidney HEK293 cells were cultured in RPMI1640 medium (ATCC, Manassas, VA, USA). MDA-MB-231 (mutant KRAS) TNBC cells were grown in Dulbecco’s modified Eagle’s medium (Corning, Manassas, VA, USA). PC-3 prostate cancer cells were grown in F-12K medium (ATCC). MCF-10A cells were cultured in MEGM medium (Lonza, Walkersville, MD, USA). Media were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cell authentication was performed by short tandem repeat analysis. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells stably expressing a control scrambled shRNA (CshRNA), MUC1shRNA, ZEB1shRNA, DNMT3bshRNA, empty vector or MUC1-C were generated as described (30 (link)–32 (link)). Cells were transfected to express a control siRNA (AM4611; ThermoFisher Scientific, Waltham, MA, USA) or RASSF1A siRNA (AM16708; ThermoFisher Scientific) in the presence of Lipofectamine RNAimax reagent (Invitrogen, Carlsbad, CA, USA).