SOCS expression by a ligand-independent inducible approach alters CXCL12-mediated mobilization of Sca-1⁺c-Kit⁺Lin^–cells. (A) pLVTHM-tetO/SOCS1 (top panels, lanes 3-4)–, tetO/SOCS3 (bottom panels, lanes 3-4)–, or tetO/mock (lanes 1-2)–transduced Lin^– cells, untreated (lanes 1,3) or tetracycline treated (lanes 2,4), were lysed and analyzed in Western blot with anti-SOCS1 or -SOCS3 antibodies, as indicated. As control, membranes were developed with anti–β-actin mAb. (B) Cells as in panel A, untreated or tetracycline treated, were allowed to migrate toward a CXCL12 gradient. The migration index was calculated as a percentage of input cells. Data represent the mean ± SD of triplicate determinations. (C) Flow cytometry analysis of transduced Lin^– cells in PBLs and BM of mice, untreated or doxycycline pretreated (48 hours), intrafemorally reconstituted with cells as in panel A. The cells are tracked by detection of dsRed/GFP-positive cells. Values represent the percentage of positive cells. Histograms correspond to representative animals (n = 8). (D) Flow cytometry analysis of Sca-1⁺c-Kit⁺Lin^– cells in periphery (right) and in BM (left), untreated or doxycycline treated (15 days after reconstitution), intrafemorally reconstituted with cells as in panel A. Cells are tracked by detection of dsRed/GFP-positive cells and represented as a percentage of Sca-1⁺c-Kit⁺Lin^– dsRed/GFP-positive cells versus total cells detected; **P ≤ .01; ***P ≤ .001. (E) Adhesion of cells as in panel A, untreated or tetracycline treated, to the stromal cell line MS-5. Results are expressed as a percentage of maximum adhered cells. Data represent the mean ± SD of quadruplicate determinations; ***P ≤ .001. (F) Cells as in panel A, untreated or tetracycline treated, were plated on confluent MS-5 or FBMD-1 monolayers in 96-well plates. We calculated the mean frequency ± SD CAFCs at culture day 28, using Poisson statistics; the data represent 1 of 2 independent experiments with similar results; *P ≤ .05,**P ≤ .01.