Depletion of IL-8 in PC-3 and DU145 cells by siRNA transfection: A and C: Decrease in IL-8 mRNA levels: Cells were transfected with 50 nM of control-siRNA (C-siRNA) or Smartpool siRNA against IL-8 (IL-8 siRNA). Total RNA was extracted 48 h after transfection to determine the levels of IL-8 mRNA by Q-RT PCR, as described in Materials and Methods (text). Each RNA sample was also simultaneously subjected to Q-RT PCR to determine the level of GAPDH mRNA for normalization. The threshold cycle (Ct) of each reaction generated by the iCycler program (Bio-Rad Inc.) was used to determine the relative mRNA levels in the test samples, using the formula: Fold Difference (FD) = 1-(2ΔCt), where, ΔCt = (Cttest RNA - CtGAPDHRNA). Final value of IL-8 mRNA, relative to that of GAPDH was expressed as: 1/FD × 100 to obtain integer values, in arbitrary units. B and D: Levels of IL-8 protein secreted into the culture medium, 48 h after transfection. IL-8 levels were measured using an ELISA kit. Levels are expressed as % of untreated control. Untreated PC-3 cells secreted ~ 60 ng IL-8/106 PC-3 cells/24 h and ~ 10 ng IL-8/106 DU145 cells/24 h.