Oncogenic PIK3CA Expression in ARK-2 Cells

Full-length, wild-type and mutated (R93Q and H1047R mutations of PIK3CA p110, (a kind gift from Dr Daphne Bell, NIH) (Rudd et al, 2011 (link)) cDNA were cloned into the pFastBac vector (Invitrogen, Waltham, MA, USA). Briefly, the Escherichia coli strain BL21 (DE3) (Agilent Technologies, Santa Clara, CA, USA) was transformed with these vectors. The transformed cells were grown in LB broth containing 100 μg ml⁻¹ ampicillin. After expansion, the plasmid DNA was purified by the QIAGEN (Valencia, CA, USA) plasmid kit according to the manufacturer instruction. ARK-2 cells were then transfected with oncogenic (activating) PIK3CA constructs using the FuGENE-6 transfection reagent (Roche, South San Francisco, CA, USA), according to the manufacturer's protocol. In a similar fashion, ARK-2 cells were transfected with empty plasmids with the same protocol. Stably transfected cells were selected in the presence of 600 mg ml⁻¹ G418 (Invitrogen).