Simultaneous Gene Silencing for Apoptosis

All cell lines were seeded at a density between 3–5 × 10⁴ cells/well and were transfected with siRNAs using Oligofectamine (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. In each transfection 72 nM of siRNAs were used in total. 12 nM of siRNA were applied per target gene, filled up with nonsense siRNA, in low-dose and combined transfections for simultaneous gene silencing (SGS). The target sequences shown in Table S3 were used with 3′-dTdT overhangs. Regarding BCLX and MCL1, the siRNAs target only the transcripts which code for the long forms of the proteins with anti-apoptotic functions [13 (link)]. As negative control we used Allstars siRNA (Qiagen, Hilden, Germany), as positive control siRNA targeting KIF11/Eg5, an essential cytoskeletal component. After incubation for 72 h the cells were harvested for flow cytometry and a caspase activity assay to determine the apoptosis rate. Gene silencing was confirmed by qRT-PCR and Western blot.