Ciap2

MEFs were seeded at 150,000 cells/well of a 6 well plate. The next day cells were washed with PBS and media without antibiotics was added. Cells were transfected with siRNA’s complexed with Lipofectamine RNAiMAX according to manufacturers instructions. Briefly, 7.5 µL of 20 µM stock stealth siRNA oligo was added to 150 µL of optimem. Nine microlitre of RNAiMAX was added to another 150 µL of optimum. The tubes were mixed together before incubating at room temperature for 5 min. 250 µL of this was then added to the cells and incubated overnight. Cells were seeded at 50,000 cells/well of an 8 chamber μ-slide (Ibidi) and allowed to plate down overnight before microscopy was performed as described above. The following stealth siRNAs were used from Thermo Fisher Scientific; cIAP1 (MSS273215), cIAP2 (MSS202113) and XIAP (MSS202115). Activity of each siRNA was confirmed using qPCR with SYBR Select Master Mix (applied biosystems # 4472908) and the following primers: cIAP1 (Forward 5′-GAAGAAAATGCTGACCCTACAGA-3′, Reverse 5′-CATGACGACATCTTCCGA-3′), cIAP2 (Forward 5′- TCGATGCAGAAGACGAGA-3′ Reverse 5′-TTTGTTCTTCCGGATTAGTGC-3′, XIAP (Forward 5′-GCTTGCAAGAGCTGGATTTT-3′, Reverse 5′-TGGCTTCCAATCCGTGAG-3′). Actin was used as a reference gene. PCR was done in 384 well plates in a 7900HT Fast Real-Time PCR System.