On target plus simcl 1

siRNA transfections were performed on 3 × 10⁵ to 4 × 10⁵ cells in 35-mm culture dishes with antibiotic-free McCoy's 5A medium supplemented with 10% FBS and DharmaFECT2 transfection reagent (Dharmacon, Inc., no. T-2002-01). On-Target Plus siMcl-1, siBcl-xL, or siControl (Dharmacon, Inc., nos. L-004501-00, L-003458-00, and D-001810-10-05) was suspended in 1× siRNA buffer (Dharmacon, Inc., no. B-002000-UB-100) and added to a plate at a final concentration of 22 nM. Cells transfected with either siBcl-xL or siMcl-1 individually were harvested 48–72 h after transfection for Western blot analysis. Double siRNA knockdown (Bcl-xL and Mcl-1) was performed sequentially. Cells were first transfected with siBcl-xL or siControl. Forty-eight hours later, cells were split 1:2, with one half transfected with Mcl-1 siRNA and the other with siControl. Twenty-four hours later, cells from the double-knockdown plates and the siControl plates were harvested for Western blot analysis with PARP antibody or stained by Hoechst dye for quantification of the condensed nuclei.