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. 2014 Jul 27;1(7):540–555. doi: 10.18632/oncoscience.70

Figure 2. Flow-cytometric analysis of replication after MMC reveals mild replication inhibition in primary human cells.

Figure 2

A) An experimental design. Cells were pulse-labeled with EdU (60 min), treated with MMC (25μM /1hr) or vehicle (DMSO), and allowed to recover, then pulse-labeled with BrdU (60 min). Note that time stamps used throughout this Figure refer to the interval between the end of EdU pulse and the start of BrdU pulse, as shown in (A). All samples were harvested at the same time at the end of the experiment. B) Example of flow-cytometric data. Shown are dot plots of HFK4 cells depleted of FANCD2. Cells were stained for EdU (Y axis) and BrdU (X axis). Dotted lines mark the quadrants for positions of four possible cell populations as defined by their EdU and BrdU staining levels. EdU+/BrdU+ cells (upper right quadrant) are cells that were treated by MMC (or DMSO) in an ongoing S phase and that continue to replicate DNA after MMC. EdU-/BrdU+ cells (lower right quadrant) are cells that were treated by MMC (or DMSO) outside of S phase and are entering S phase after MMC. EdU+/BrdU-cells (upper left quadrant) are cells that were treated in S phase and have now exited S phase or stopped replicating. An asterisk in an upper left panel marks position of residual calibration beads. C) Declines in percentages of EdU+/BrdU+ (ongoing S phase) cells over time were derived from dot plots as in (B) and plotted. X axis represents hours lapsed between EdU and BrdU pulses. Lines represent trendlines derived from the data. Shown is a representative experiment performed with HFK4 cells. D) Flow cytometry profiles of BrdU incorporation levels in EdU+ HFK4 cells from a representative experiment. Indicated times are hours lapsed between EdU and BrdU pulses. E) Percentages of EdU-/BrdU+ (new S phase) cells at indicated times between pulses after MMC (or DMSO) treatment were derived from dot plots as in (B) and plotted as bar graphs. Two independent experiments performed with control and FANCD2-depleted, HFF4 and HFK4 cells were averaged. Controls are non-specific shRNA (shNS) expressing cells for HFF4s and empty vector-expressing cells for HFK4s. Error bars are standard deviations.