Figure 4

Cell division and gene expression in U2OS, AGN2a and SCCVII cells. Tumor cells were labeled with CFSE to detect changes in fluorescence that occur with cell division post-nucloefection. (A) U2OS, (B) cultured AGN2a, (C) SCCVII cells were analyzed for CFSE fluorescence by flow cytometry immediately following nucleofection or cultured up to 3 days following nucleofection. The solid gray peak (far left of each panel) represents unstained cells. Peaks decreasing in fluorescence (right to left) indicate the loss of CFSE fluorescence over time as cells divide. Cells were analyzed at hour 2, 4, 6, 8, 10, day 1, day 2, and day 3. Only U2OS showed peaks indicating decrease in fluorescence prior to day one: at hour 2, and hours 4–10. D) In parallel experiments, the average percent of cells expressing RFP from 3 separate experiments at the indicated time points following nucleofection was determined in U2OS (■), SCCVII (▲) and AGN2a (◆) cell lines by flow cytometry. E) Average percentage of cells expressing CD137L at time 0, 2 hours, and 6 hours post-Nucleofection in U2OS and SCCVII cells. The error bars represent the standard deviation from 3 experiments. F) Average viable cell number of U2OS, SCCVII and AGN2a tumor cells post-nucleofection from 3 separate experiments, expressed as a percentage of the number of cells originally nucleofected.