Figure 3

Confocal images of FITC-labeled plasmid in U2OS cells immediately and 3 days following nucleofection. A) Confocal images captured immediately following nucleofection: a) fluorescence of FITC-labeled pUC19 plasmid, b) phase contrast image, c) TOTO3 stained nuclei (dark blue), d) overlay of the images in (a) and (c) showing FITC-labeled plasmid in the nucleus. B) Confocal images captured 3 days following nucleofection: a) fluorescence of FITC-labeled pDSRed2C-1 plasmid, b) fluorescence from expression of RFP encoded on the FITC-labeled pDSRed2C-1 plasmid, c) TOTO3 stained nuclei, d) an overlay of the images in a, b and c showing FITC-labeled plasmid, expression of RFP and nuclear localization. C) Average number of plasmid copies per cell as determined by real-time PCR of the plasmid encoded neo gene from nuclear DNA harvested immediately following nucleofection. D) Plasmid neo copies amplified from nuclear DNA harvested 3 days following nucleofection. In both C and D the gray bars represent neo amplification from U2OS cells nucleofected with 0.5 μg pDSRed2C-1 per 10⁶ cells and the black bars represent neo amplification from non-nucleofected U2OS cells. Nuclear DNA was harvested and amplified from 3 separate samples, error bars indicate the standard deviation from the average of triplicate samples.