HGF-induced increase in CXCR4 expression depends on PKCζ activity.

<p>(A). Time course of relative p-PKCζ levels as determined by immunoblot in MDA-MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. The experiment was repeated three times with similar results. A representative study is shown. (B) and (C). 100 µM PKCζ-siRNA1 (si1) or PKCζ-siRNA2 (si2) transient transfections and siRNA-mediated PKCζ protein silencing in MDA-MB-436 cells. p-CXCR4 and p-PKCζ expression levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF or/and 100 µM PKCζ-siRNA1 (si1) or PKCζ-siRNA2 (si2). The experiment was repeated three times with similar results. A representative study is shown. (D). Western blot analysis of total CXCR4 expression and p-CXCR4 levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF, 2 µM cholesterol sulfate (Choles sul), or PBS. As indicated, 10 µM PS of PKCζ (PSζ), PKCε (PSε), or PKCα/β (PSα/β) or 25 µM NSC23766 (Rac1 inhibitor, Rac1 inh) was used. The experiment was repeated three times with similar results. A representative study is shown. (E). Membrane CXCR4 expression in MDA-MB-436 cells incubated for 24 hours with 50 ng/ml HGF, 2 µM Choles Sulfate, or PBS. Where indicated, 10 µM PS of PSζ, PSε, or PSα/β was used. The flow cytometry analysis data are shown in arbitrary units (AU) normalized to PBS as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (F). Western blot analysis of Rac1-GTP and total-Rac1 in MDA-MB-436 and MCF-7 cells treated with 50 ng/ml HGF with or without 25 µM NSC23766. The experiment was repeated three times with similar results. A representative study is shown. (G). Dose-dependent inhibition of HGF-induced p-PKCζ was achieved using NSC23766 in HGF-treated MDA-MB-436 cells; the cells were assayed by western blot. The experiment was repeated three times with similar results. A representative study is shown. (H). Western blot analysis of Rac1, PKCζ and CXCR4 expression in MDA-MB-436 cells cultured for 48 hours with 100 µM Rac1-siRNA. The experiment was repeated three times with similar results. A representative study is shown. (I). Cellular distribution of PKCζ (left panel) and CXCR4 (right panel) before and after HGF (50 ng/ml) or NSC23766 (50 µM) stimulation for 10 minutes. Representative of two independent experiments was shown.