Rapamycin blocks CXCL12 induced migration and actin polymerization of T cells.

<p>(A) Primary human T cells were labeled with calcein (5 µM) for 1 h in media and washed. Pretreatment of labeled cells was done with or without rapamaycin (100 ng/ml) for 1 h. Cells (1×10⁵ in 100 µl) were placed in the upper wells of 24-well transwell migration chambers with 5 µm pores (Corning, Corning, NY). In the lower wells, either medium alone or CXCL12 (100 ng/ml) was added to a total volume of 600 µl, and the chambers were incubated for 2 hours at 37°C in 5% CO2 incubator. Triplicate well determinations were performed for each treatment. The level of fluorescence of cells migrating across the chamber was assessed using a microfluorimeter. * indicates the value is statistically significant at p<0.05 level (n = 4). PMI indicates ‘percent migration index’. (B) Primary human T cells were treated with CXCL12 for 30 minutes in the presence or absence of pretreated rapamycin. Confocal microscopy was done according to the procedure mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a> to monitor actin polymerization. Red color indicates the actin polymerization. Representative images are shown from each treatment group. (C) Effect of rapamycin and KU-0063794 (KU) on CXCL12-induced cell migration, and effect of MIP3β and TARC in the presence or absence of rapamycin on the migration of resting T cells were performed as described in panel A. (D) Migration assay for CEM cells was performed similarly as primary T cells described above. (E) Dose response curve for rapamycin effect.</p

Role of mTOR in CXCL12-induced signaling and cell migration.

<p>(A) Whole cell lysates from CEM stable clones carrying either empty vector (EV) or shRNA constructs (sh-222 and sh-224) were analyzed by western blot analysis to determine the levels of total p70^S6K1. (B) Whole cell lysates from EV and sh-222 clones treated with CXCL12 for 30 minutes were analyzed by western blot analysis to determine the levels of phospho-p70^S6K1. (C) CEM cells were pretreated with rapamycin (100 ng/ml) and KU-0063794 (1 µM) for 1 hour, and then CXCL12 treatment was done for 30 minutes. Equal amounts of whole cell lysates were analyzed by western blot analysis. (D) Migration assay for EV, sh-222 and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1A</a>. (E) Surface expression of CXCR4 for EV, sh-222, and sh-224 were determined by FACS analysis, and the levels are expressed as mean fluorescence intensity (MFI). (F) Confocal microscopy to monitor CXCL12-induced actin polymerization in EV, sh-222, and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1B</a>. (G) In vivo migration of EV and sh-222 clone mediated by CXCL12 was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a>. Tissue staining from individual mice is shown here.</p