Journal: Scientific Reports

Article Title: Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

doi: 10.1038/srep03759

Figure Lengend Snippet: 20 × 10 3 A431 cells were mixed with 80 × 10 3 HeLa cells or 80 × 10 3 MCF7 cells, and the mixture was transduced with either His-tag affinity purified AAV-2_Affibody, AAV-2_DARPin or AAV-2 ΔHSPG capsids delivering the cytosine deaminase. Cells only served as a negative control and A431 cells incubated with 20 μM camptothecin for 4 h served as a positive control for apoptotic cells. After addition of the viral particles, cells were incubated in DMEM containing 250 μM 5-FC. 48 h later, cells were detached, washed and incubated with an anti-EGFR-Alexa488 antibody on ice for 1 h. An anti-Annexin-V-PE antibody was added and cells were analyzed by flow cytometry. For every sample 30,000 events were recorded. Dead cells were excluded using initial gating based on forward scatter height versus sideward scatter height. The anti-Annexin-V fluorescence is plotted against the EGFR fluorescence and the percentages of cells in each quadrant are given below each plot. Experiments show that EGFR expression of A431 cells decreases upon apoptosis (right panel). rAAV without targeting does not mediate prodrug activated apoptosis (middle panels). Targeted AAV primarily induces EGFR dependent apoptosis, but a bystander effect might harm nearby cells.

Article Snippet: Flow cytometry analysis with simultaneous staining for EGFR expression and Annexin V revealed specific apoptosis induction in EGF receptor overexpressing A431 cells with comparable efficacies for AAV2-Affibody and AAV-2-DARPin ( , left panels).

Techniques: Transduction, Affinity Purification, Negative Control, Incubation, Positive Control, Flow Cytometry, Fluorescence, Expressing