Functionally associated targets in mantle cell lymphoma as defined by DNA microarrays and RNA interference by Eva Ortega-Paino, Johan Fransson, Sara Ek, and Carl A. K. Borrebaeck Blood Volume 111(3):1617-1624 February 1, 2008 ©2008 by American Society of Hematology

Gene expression profile of MCL-associated genes. Gene expression profile of MCL-associated genes. A total of 46 MCL-associated genes were identified by filtering the transcriptionally profiled genomes. The relative expression of selected genes is shown for the 21 MCL samples, 5 B-cell populations, and the 3 different MCL cell lines (NCEB1, Granta 519, and SP53). Hierarchic clustering using bootstrapping (n = 100) was performed, and the 5 B-cell populations were shown to be significantly (P < .05) separated from the MCL samples and the tumor cell lines. Eva Ortega-Paino et al. Blood 2008;111:1617-1624 ©2008 by American Society of Hematology

Experimental Controls. 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. Eva Ortega-Paino et al. Blood 2008;111:1617-1624 ©2008 by American Society of Hematology

Assessment of proliferation after silencing of selected targets. Assessment of proliferation after silencing of selected targets. A total of 2 unique siRNA sequences were designed and synthesized against each of 20 selected transcripts derived from the DNA microarray study. Granta 519 cells were nucleofected with the siRNAs and appropriate control siRNAs, and the proliferative effect of the sequence-specific knockdown was assessed. Electroporation control (ie, electroporation without any siRNA) was set to 100%. Bars represent plus or minus SD of triplicate samples. Eva Ortega-Paino et al. Blood 2008;111:1617-1624 ©2008 by American Society of Hematology

Effect of HDGFRP3 siRNAs on different MCL cell lines. Effect of HDGFRP3 siRNAs on different MCL cell lines. A total of 6 MCL cell lines were electroporated with 100 pmol of a pool of siRNAs targeting the HDGFRP3 (□), negative control siRNA (▩), or Eg5+ control (■). At 72 hours after transfection, the proliferation of each cell line was determined. Eva Ortega-Paino et al. Blood 2008;111:1617-1624 ©2008 by American Society of Hematology

Evaluation of gene expression. Evaluation of gene expression. Gene expression was evaluated after silencing the selected targets in Granta 519. Evaluation of mRNA levels was performed at 48 hours after electroporation. Error bars are plus or minus standard deviation of 3 independent experiments. Eva Ortega-Paino et al. Blood 2008;111:1617-1624 ©2008 by American Society of Hematology