Reconstitution of PTEN in Jurkat cells down-regulated CXCR4-mediated chemotaxis. (A) Doxycycline (dox) induced PTEN expression in PIJ-17 but not in Col-18 or Tet-on Jurkat cells. PIJ-17, Con-18, and Tet-on Jurkat cells were treated with 1 μg/mL dox for 48 hours and the expression of PTEN (top panel) and Ser473-phosphorylated AKT (middle panel) was measured by Western blotting. The samples were also blotted with anti-ERK1/2 as loading control (bottom panel). (B) Expression of PTEN down-regulated SDF-1α–mediated chemotaxis. PIJ-17 and Con-18 Jurkat cells were cultured with or without the presence of dox (1 μg/mL) for 48 hours and chemotaxis was carried out as in Figure 1. The results were representative of at least 5 independent experiments. Data shown are means ± SD. (C-D) Time course of PTEN expression under dox induction and its effects on chemotaxis. PIJ-17 Jurkat cells were treated with 1 μg/mL dox for the indicated periods of time, and the levels of PTEN and Ser473-phosphorylated AKT were detected by Western blotting (C). The same cells were also assayed for chemotactic responses (D). Data shown are means ± SD. The results were representative of 3 independent experiments. (E) PTEN expression had no effects on CXCR4 surface expression. PIJ-17 Jurkat T cells were cultured with or without dox (1 μg/mL) for 48 hours and CXCR4 expression were detected by flow cytometry. The results were representative of 2 independent experiments. The histograms show cell-surface staining with anti-hCXCR4 (solid line) or isotype-matched human IgG2a (dashed line).