Table 2.

A. BAG4, LSM1, and C8orf4 are preferentially present in insulin-independent and EGF-independent clones following a multiple gene infection strategy
GeneInsulin-independent clones with insertEGF-independent clones with insert
B. BAG4, LSM1, and C8orf4 cooperate to induce EGF-independent growth
Selection conditionsGene insert
Empty vectorLSM1C8orf4BAG4LSM1 + C8orf4LSM1 + BAG4C8orf4 + BAG4LSM1 + C8orf4 + BAG4
+BL, −Insulin+++++++
+BL, −EGF++++
+BL, −Insulin, −EGF
  • NOTE: Lentiviral expression constructs were created for candidate oncogenes from the 8p11-p12 amplicon, including LSM1, EIF4EBP1, C8orf4, PPAPDC1B, ZNF703, RAB11FIP1, BAG4, and FGFR1. 293FT cells were transfected with each lentiviral vector, and virus was harvested 48 hours after transfection. Virus of the same titer from all eight genes was combined and used to infect MCF-10A human mammary epithelial cells. To select for the presence of insert(s), 10 μg/mL blasticidin was added to MCF-10A cells 48 hours after infection, as well as media without insulin or without EGF to determine gene combinations that lead to growth factor independence. A, RNA was isolated from 20 clones growing in insulin-independent medium resulting from four separate infections or from 10 clones growing in EGF-independent medium resulting from three separate infections. The presence of various inserts was evaluated by RT-PCR using insert-specific primers. B, MCF-10A cells were infected with C8orf4, BAG4, or LSM1 lentivirus individually, two at a time, or in a combination of three. Selection medium with 10 μg/mL blasticidin and without insulin, EGF, or both was added 48 hours after infection. −, no colony growth; +, colony growth under the given conditions.

    Abbreviations: BL, blasticidin; −EGF, EGF independent; −Insulin, insulin independent.