Activation of the SDF-1/CXCR4 pathway in Tesi cells. (A) Tesi, Tesi-Tax control (Tesi cells cultivated for 10 days in the presence of 1 μg tetracycline for Tax-1 suppression), and Tesi-PTX (Tesi cells cultivated for 24 hours in the presence of 100 ng/mL pertussis toxin) cells were stimulated for 5 minutes with 30 ng/mL SDF-1, 30 ng/mL MCP-1, or 50 ng/mL RANTES. Cells were lysed, and ERK1/2 activation was measured by Western blotting with an anti–phospho-p42/44 monoclonal antibody (top). The same blot was stripped, and immunoblotting was performed with an anti–Tax-1 antibody (bottom). Arrows indicate p42, p44, and protein bands. (B) Tesi and Tesi-Tax control cells were loaded with 5 μM Fura-2 for 30 minutes at 37°C in the dark. Loaded cells were washed twice, resuspended at 10⁶ cells/mL, and kept for 30 minutes at 4°C in the dark. Ca²⁺ mobilization in response to 50 nM SDF-1 was measured using a luminescence spectrometer (LS50B; Perkin Elmer) by recording the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm. Arrows indicate the injection of SDF-1. (C) Tesi and Tesi-Tax control migration assays were performed using Transwell culture chambers (5-μm pore size). Lower wells were filled with 500 μL medium (RPMI 1640, 1% FCS) containing 30 ng/mL SDF-1, 50 ng/mL RANTES, or 30 ng/mL MCP-1. Tesi or Tesi-Tax control cells (10⁵ cells) suspended in 100 μL medium were loaded into the upper wells. After incubation for 2 hours at 37°C, the cells that had migrated to the lower wells were counted and are shown as percentages of the input cells. Data are reported as mean ± SD of 3 independent experiments in triplicate.