External IL-8 restores the functions of IL-8 depleted by siRNA transfection: Effect of addition of recombinant human IL-8 (rhIL-8) on Cyclin D1 level was determined by western blotting. PC-3 cells transfected with 50 nM C-siRNA or IL-8 siRNA and cultured in complete medium for 48 h were stimulated with rhIL-8 (25 ng/ml) and harvested by SDS-sample buffer at 0, 15, 30 and 60 min, and probed for Cyclin D1 and β-actin (A (i)). Expression of Cyclin D1 relative to β-actin in PC-3 cells: Cyclin D1 was quantified by densitometry and expressed as a ratio of the band intensity with respect to β-actin (A (ii)). Note that Cyclin D1 level rose significantly in cultures depleted with IL-8 but supplemented with external IL-8. B. IL-8 stimulates Cyclin D1 in LNCaP cells. LNCaP cells cultured in complete medium for 48 h were stimulated with rhIL-8 before harvesting and determining Cyclin D1 level, as described for A. C. Cyclin D1 in IL-8 stimulated LAPC4 and LAPC4IL-8 cells. (i). The experimental conditions to determine Cyclin D1 were identical to the one used in B, except that LAPC4IL-8 cells were used that constitutively produce IL-8 (8 ng/106 cells/24 h) . Note that, while significant increase in Cylcin D1 is observed in LAPC-4 cultures stimulated with IL-8, the increase in Cyclin D1 level was negligible in LAPC4-IL-8 cells (C(ii)). D. Phosphorylated ERK1/2 levels are lower in IL-8 depleted cells: Levels were determined by an ELISA from cell lysates prepared from EGF-stimulated PC-3 cells with C-siRNA or IL-8 siRNA transfection. Error bars indicated are Mean ± SD from pooled data of two independent determination with triplicate samples. Significance of the observation was tested using Student's t-test as described in Methods. (* indicates p = 0.05, determined using t-test as described in the Methods.).