Transfection of Cell Lines with Antisense Oligonucleotides

Cells (HeLa, A549, MCF-7 and MDA-MB-231 obtained from ATCC) were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 units ml⁻¹ penicillin and 10 μg ml⁻¹ streptomycin at 37 °C, and 5% CO₂. All cell lines were tested for mycoplasma contamination and were not authenticated. U1, U2 antisense, and control morpholino oligonucleotide sequences were 5ʹ-GGTATCTCCCCTGCCAGGTAAGTAT-3ʹ, 5ʹ-CCTCTTACCTCAGTTACAATTTATA-3ʹ and 5ʹ-TGATAAGAACAGATACTACACTTGA-3ʹ, respectively^{8 (link)}. Oligonucleotides were transfected into 1 × 10⁶ cells using a Neon transfection system (Invitrogen) according to the manufacturer’s instruction to achieve the desired final doses indicated in the text and figures.