Overexpression of GRK6 inhibits CXCR4 signaling and cell migration. (A) Left panel: Lentiviral FUW-Cherry (control) or FUW-Cherry-GRK6 (FUW-GRK6)- infected Daoy cells were serum-starved and treated with AMD3100, an antagonist of CXCR4, for 24 h prior to CXCL12 (SDF-1α) stimulation (100 ng/ml for 15 min). The results show that overexpression of GRK6 inhibits CXCL12-induced P-ERK and CXCL12-induced P-ERK is completely abolished by AMD3100 at 2.5 μg/ml in Daoy-FUW-GRK6 cells, not in Daoy-FUW-Cherry cells, that potentiates the CXCR4 inhibitory effect of AMD3100. GRK5 serves as internal control (no change) to demonstrate that effects are GRK6-specific; Right panel: Quantitative analysis of Western blot shows CXCL12-induced P-ERK is significantly inhibited by AMD3100 treatment in Daoy with overexpression of GRK6 [P < 0.05, lane 3, 0.59 ± 0.1 vs. lane 6, 0.31 ± 0.03, the ratio of P-ERK/ERK in lane 1 is equal to 1.00 (100%), three independent experiments]. (B) Boyden chamber migration assay shows that serum-mediated migration is significantly decreased in cells with FUW-GRK6 overexpression (P < 0.01), compared to FUW-Cherry control cells. (C) xCELLigence was used to monitor real-time cell migration and proliferation. In the left panel, the results show that serum-mediated cell migration is significantly decreased in the cells with FUW-GRK6 overexpression (the upper two lines represent the migration of either FUW-Cherry or FUW-GRK6 in serum-containing medium. The bottom two lines represent the migration of either FUW-Cherry or FUW-GRK6 in serum-free medium). In the right panel, the results show that overexpression of GRK6 has little effect on cell proliferation either at high cell density (2 × 104/well, 20 K) or low cell density (5 × 103/well, 5 K).