Tumor/Endothelium Crosstalk Attracts T Cells Figure 5. The primary medulloblastoma chemokine signal triggering the release of RANTES is mediated by MIF. (A) Increasing concentrations of exogenous recombinant human (rh)MIF added to 72 hour cultures of bEND.3 cells induced the release of murine RANTES in a rhMIF-dose-dependent manner. (B) Supernatants from cell cultures analyzed for the production of MIF showing that Daoy cells transduced with a MIF-specific shRNA encoding construct (MIF-KD Daoy) secreted substantially less MIF (,20%) compared to the parental Daoy cell line. (C) RNA was extracted from tumors established from Daoy, MIF-KD Daoy and IL-8-KD Daoy and analyzed for the presence of RANTES as well as GAPDH and b-Actin (controls). While IL-8-KD Daoy tumors showed minimal inhibition of RANTES RNA (4.8 fold decrease), MIF-KD Daoy tumor RNA showed a 38 fold decrease in expression compared to the parental cell line Daoy. Both house-keeping genes were minimally altered. (D) MIF-KD Daoy cells induced similar size tumors in comparison to parental Daoy cells but failed to induce T lymphocyte migration in vivo. doi:10.1371/journal.pone.0020267.g005 PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20267