Figure 4

Decrease in survival and angiogenic factors in IL-8 depleted AIPC cells: (A). IL-8 depletion reduced the steady state levels of activated AKT (Phospho-AKT). Phospho-AKT levels were detected by western blotting in cell lysates, prepared 48 h following C-siRNA or IL-8 siRNA transfection of PC-3 or DU145 cells. Numbers at the bottom of the gel indicate p-AKT band intensity relative to that of C-siRNA transfectants, after normalizing the band intensities to that of T-AKT. (B). NFkB activity in PC-3 cells measured using a NFkB-Luciferase reporter construct co-transfected with control or IL-8 siRNA and TK renilla plasmid as described in the text. (C). VEGF levels in IL-8siRNA or C-siRNA transfected PC-3 and DU145 cells. Relative levels of VEGF mRNA, 48 h after transfection, were determined by Q-RTPCR. VEGF protein levels in culture-conditioned medium of transfected cells from 24 h-48 h, following transfection, were determined using an ELISA kit (R&D system Inc.) * p < 0.05. D. Decrease in the invasion and motility activity of PC-3 and DU145 cells transfected with IL-8 siRNA. Fractions of cells migrated to bottom wells either due to chemotaxis or invaded the Matrigel-coated filters were determined by MTT assay, as described in the text. The data shown is from a single experiment with triplicate wells, similar results were obtained from two independent experiments. Note the %-invaded cells is higher than that of % motility because, the invasion assay was terminated at 48 h and motility assay at 24 h. At 24 h, MTT assay was too insensitive to estimate of %-invaded cells in DU145 cells. Error bars: Mean ± SD. * represents the probability (p) that the measured values presented from IL-8siRNA and C-siRNA transfectants are the same; p < 0.001 in all experiments (B-D).