Effects of Dot1L depletion on cell cycle progression.

<p>HCT116 cells transfected with siRNA directed against <i>DOT1L</i> or scrambled siRNA control three times, first with a 48 h interval followed by a 72 hr interval. Cells were collected for cell cycle analysis by FACS 3 days after the third transfection. EdU were added to cells for 45 minutes before harvesting cells. Click-iT EdU kit from Invitrogen was used to detect replicating cells and DAPI was used to determine DNA content. A. The left panel shows a representative cell cycle profile for cells transfected with control siRNA. The middle panel shows a representative cell cycle profile for cells transfected with siRNA directed against DOT1L. The right panel shows a histogram illustrating the fraction of cells in early, middle and late S-phase (ES, MS and LS, representing the number of cells in the P2, P3 and P4 FACS gates, respectively). Two stars represent a statistically significant change with p value lower than 0.01; three stars represent a statistically significant change with p value lower than 0.001. Dot1L depletion increased the fraction of late S-phase cells. B. The left and middle panels show the cell cycle distribution of the same cells as in A, illustrating the FACS gates used to identify the sub-G1, non-replicating S and >G2/M cell populations. The histogram plots the fraction of the gated populations in DOT1L depleted cells divided by the fraction of the same gated populations in cells transfected with a control siRNA. A star represents a statistically significant change with p value lower than 0.05; two stars represent a statistically significant change with p value lower than 0.01. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542.s009" target="_blank">Table S2</a> shows the fraction of cells at each cell cycle phase. Dot1L depletion caused a limited increase in the fraction of apoptotic cells, cells with S-phase DNA content that did not incorporate EdU and cells with DNA content greater than G2/M.