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. 2014 Oct 22;94(4):945–962. doi: 10.1111/mmi.12812

Figure 6.

Figure 6

Depletion of cyclin 6 and Spt16 results in a comparable accumulation of cells at the G2/M cell cycle stage (4n DNA content), but only depletion of Spt16 leads to derepression of a silent VSG ES promoter.A. Knockdown of cyclin 6 does not result in significant derepression of silent ES promoters. Cyc6 RNAi was performed in the T. brucei RYT3 reporter cell line (Hughes et al., 2007), which has a GFP gene immediately downstream of the promoter of the silent VSG221 ES. Cells were harvested after induction of Cyc6 RNAi with tetracycline (Tet) for the time indicated in hours (h). Cells were stained with propidium iodide (PI) and subjected to flow cytometry. Derepression of GFP behind the silent VSG221 ES promoter was monitored in the FL1 channel (x-axis), with values over 20 considered significant ES promoter derepression. The DNA content was monitored in the FL2 channel (y-axis) with the G2 gate indicating cells with 4n DNA content. The degree of ES promoter derepression in cells stalled in G2/M is shown, with the values corresponding to the mean of three independent experiments with the standard deviation.B. Knockdown of Spt16 results in significant derepression of ES promoters specifically at the G2/M cell cycle stage. Further the experiments in this panel are as described above.