Figure 3

Stable modification of ADSCs and melanoma B16f10 cells with the use of DNA integrating vectors. A) Flow cytometric analysis of EGFP expression by ADSCs. (Left plot) Mean percentage of EGFP-expressing ADSCs post 48 h of nucleofection with pmaxGFP vector, indicating efficient transgene transfer into mouse ADSCs. (Right plot) Mean percentage of EGFP-positive ADSCs two weeks after nucleofection with pmhy GENIE-3 and hygromycin selection. Results are representatives of triplicate experiments. B) Fluorescence microscopy image of EGFP-expressing ADSCs after nucleofection with pmhy GENIE-3 and two weeks selection with hygromycin. C) Stable RFP-expressing melanoma cells after lipofection with pDsRed-attb-zeo/pCMVInt and three weeks of zeocin selection. D) EGFP-expressing melanoma cells after lipofection with pBEB/pCMVInt and three weeks of geneticin selection. E) Western blot analysis of IFNγ from ADSC cell lysates. Modified ADSCs were selected with hygromycin following nucleofection with either pmhy GENIE-3 (Lane 1) or pmhy GENIE-3-IFNγ (Lane 2). Blotting was performed using a monoclonal antibody against mouse IFNγ. F) Cytofluorimetric evaluation of TRAIL expression in ADSCs selected with geneticin following co-nucleofection with pBEB/pCMVInt (Violet histograms) and pBTB/pCMVInt (white histograms). ADSCs were stained with a PE conjugated monoclonal antibody to mouse TRAIL. A single representative experiment of triplicates is shown. (Left plot) Surface staining of TRAIL-ADSCs after 48 h of co-nucleofection. (Right plot) Surface staining of TRAIL-ADSCs after co-nucleofection and three weeks of geneticin selection. PE = phycoerythrin.