RNAi-mediated silencing of endogenous human CD40 expression. (A) Screening for effective siRNA inhibitors of endogenous CD40 expression. ECV-304 cells were transfected for 4 hours with 100 nM of the siRNAs as indicated in “Study Design.” Total cellular RNA was extracted 48 hours after transfection and analyzed by real-time semiquantitative reverse-transcription PCR (RT-PCR). Human CD40 mRNA expression levels were assessed relative to the constitutively expressed cyclophilin gene (ratio of CD40 to cyclophilin mRNA). Data are presented as means and standard error of the mean (SEM) for 3 independent experiments (^**P < .01, ^*P < .05, versus siRNA-C). The lower parts of panel A depict the evaluation of CD40 silencing at the protein level. The siRNA-transfected ECV-304 cells were stimulated with proinflammatory cytokines TNF-α (100 U/mL) and interferon-γ (IFN-γ) (1000 U/mL) 24 hours prior to protein extraction and Western immunoblotting, performed 48 hours after transfection. (B) Potency of siRNA-2. ECV-304 cells were stimulated as previously described and transfected with the indicated concentration of siRNA-2, and the CD40 receptor levels relative to the β-actin protein levels were determined by Western immunoblotting and densitometry by means of Phoretix 10 software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom). Data are shown as means and SEM from 2 independent assays. (C) Specific inhibition by siRNA-2 of CD40 expression on human endothelial cells. HUVECs were lipofected for 2 hours with 100 nM siRNA-2 or its corresponding siRNA control, msiRNA-2, complexed with Targefect as described in “Study Design,” and activated with TNF-α (100 U/mL) and IFN-γ (1000 U/mL) 16 hours prior to protein extraction. Representative Western immunoblotting was performed 48 hours after transfection. The housekeeping β-actin protein was included in the Western analyses to normalize for equal loading of the gel lanes. Relative mobility of molecular weight markers is shown in kilodaltons. (D) Predicted secondary structure of human CD40 mRNA. The predicted secondary structure of CD40 mRNA (residues 1-250 from the coding region are displayed) was determined by means of the suboptimal folding program of Michael Zucker, Mfold,¹⁶  based on the energy minimization method. The targeting locations from 5 of the 8 designed siRNAs are indicated over the optimal lowest free energy structure.