Figure 5

SiRNAs specific for DcR2 and DR5 suppress gene expression in LNCaP cells. Gene-specific siRNAs and scrambled siRNAs controls (si0) (0.5 μg) were added to the media using lipophilic transfection-enhancing reagent (lipofectamine/lipo) for 4 h, and then the cells were cultured in media containing 0.1 nM R1881. Cells were harvested after 48 or 96 h for immunoblot analyses with DcR2 (A) and DR5 (B) specific antibodies. The blots were reprobed with antibody against ß-actin to confirm equal protein loading (15 μg). All the samples were run and detected together but 96 h films were cut and pasted to remove spots from replicate cell protein extracts. Representative autoradiograph results are shown and histograms represent DcR2 (right scale: 48 h incubation; left scale: 96 h incubation) and DR5 protein levels and were normalized for ß-actin expression. (C): the number of fragmented nuclei per 1,000 cells was determined in all transfection treatment conditions (scrambled and specific siRNAs and lipofectamine) with DAPI method. The values represent the mean ± SD determined from triplicate cultures of a representative experiment from 3 independent experiments. *P < 0.005 versus the other treatment conditions.