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. Author manuscript; available in PMC: 2007 Oct 11.
Published in final edited form as: Cytokine. 2006 Mar 7;33(4):188–198. doi: 10.1016/j.cyto.2006.01.006

Fig. 1.

Fig. 1

Loss of MIF expression by d-siRNA specific for MIF. (A) Cell lysates from AGN2a and AGN2a/CD80−137L cells that were untreated (lane 1), treated with transfection reagent alone (lane 2), transfected with d-siRNA specific for MIF (lane 3), or transfected with control d-siRNA specific for GFP (lane 4) using the GeneSilencer transfection system (described in Section 2) were prepared from cells collected 60−72 h after transfection and whole cell lysates fractionated by polyacrylamide gel electrophoresis. The electrophoresed proteins were transferred to membranes and detected with anti-MIF polyclonal antibody. (B) 2 × 104 AGN2a cells treated with transfection reagent alone (mock), treated with MIF-siRNA (MIF-siRNA), treated with GFP-siRNA, or untreated, AN2a, were cultured in 1 ml of c-DMEM for 48 h, supernatant collected and MIF concentration determined by ELISA. Average concentration and standard deviation from triplicate wells is shown. Only MIF-siRNA treated tumor showed significantly lowered MIF production when compared to mock transfection (P < 0.05, Student's paired t-test, used in all figures upon F-test for variance unless otherwise noted). (C) To demonstrate functional MIF activity, peritoneal macrophages loaded in capillary tubes were cultured in media alone (cntrl) or in day 3 culture supernatants from AGN2a cells (AGN2a), AGN2a cells transfected with d-siRNA for MIF (+MIF siRNA), AGN2a cells transfected with d-siRNA for GFP (+GFP siRNA), or from AGN2a cells treated with the transfection reagents in the absence of added nucleic acid (T-cntrl). Following 24 h of incubation in these culture supernatants, macrophage-loaded capillary tubes were removed from triplicate tissue culture wells and cells that had migrated out of the capillary tubes counted. Data plotted in all figures is the average and standard deviation from triplicate assay wells unless otherwise noted. Only control and MIF siRNA-treated tumor differed significantly from mock-transfected tumor (P < 0.05). The results are representative of three experiments.