IL-8 depletion in AIPC cells causes decrease in cell proliferation and cell cycle arrest: (A). Cell viability was measured using the MTT assay, 48 h after transfection. Triplicate wells were used for each measurement, experiment repeated three times per cell line. Error bars: mean ± s.e.m, n = 3. (B). Cell cycle arrest at G1/S boundary. Percent of cells in each of the three cell cycle phase was determined by flow cytometry, 48 h after siRNA transfection. Results shown are from one transfection experiment, similar results were obtained in two separate determinations. (C). Levels of Cyclin D1 and Cyclin B1 expression: Detected by immunoblotting, as described in the text. β-actin levels, as shown, were used to normalize the protein levels of Cyclin D1 and Cyclin B1. The decrease in the levels of Cyclin D1 by IL-8 depletion was 68% in PC-3 and 52% in DU145 cells. The decrease in Cyclin B1 level was 73% in PC-3 and 41% in DU145, as determined by densitometry measurement of band intensities and normalized to β-actin levels. D. IL-8 siRNA transfection decreases growth factor (IGF-1) mediated mitogenic stimulation. Cyclin D1 in cell lysates, prepared from PC-3 cells stimulated with IGF-1, was detected by western blotting. Levels of Cyclin D1 were normalized to β-actin (Actin) levels detected by re-probing the same blots.