Experimental Controls. (A) The mantle cell lymphoma cell line Granta 519 was electroporated with 100 pmol of siRNA targeting the CD40 receptor (■) or a nonexisting transcript (negative control siRNA; □). At indicated time points, the cells were harvested and the binding of a commercial anti-CD40 antibody was assessed by means of flow cytometry. The expression of CD40 (as determined by the mean fluorescence intensity [MFI] of the viable population) on cells electroporated in the absence of siRNA was set to 1. (B) To assess the RNAi knockdown on a functional level, the proliferation of transfected cells was investigated. Granta 519 cells were electroporated with 100 pmol siRNAs and incubated for 72 hours, where after the proliferative effects of the different siRNAs was determined by thymidine incorporation. Bars represent plus or minus SD. (C) Similar to panel A, Granta cells were electroporated and 48 hours later, cells were fixed and stained with propidium iodide. The DNA content of the cells was determined in the FL-2 channel in a FACScan. Cell cycle phases were analyzed using Deau-Jett-FCX model enclosed in FlowJo software (TreeStar, Ashland, OR): green lines and black picks correspond to the different phases of the cell cycle. Black horizontal lines define the amplitude of the different picks, therefore, the percentage of the different cell cycle phases.