Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen
Figure 2
Protein kinases involved in the cell cycle.
In vitro growth and cell cycle analysis for ATR (A) and CLK1 (B). A: Upper left: growth curves showing cumulative cell counts over time following tetracycline (Tet) induction (+) or not (−) of RNAi cell line in culture. Cell densities were maintained between 105 and 106 cells ml−1. Upper right: DAPI staining. Cells were stained with DAPI and the number of nuclei (N) and kinetoplasts (K) per cell was quantified (n>200) at the time-points indicated. Lower panel: flow cytometry profiles for 50,000 propidium iodide labelled cells at 24 hr or 48 hr post-induction. The DNA content of each peak is indicated. B: legend as for A, but including in upper right panel an analysis of the cytokinesis stage of 2N2K cells following tetracycline (Tet) induction (+) or not (−) of CLK1 RNAi cell line in culture. Lower left panels: flow cytometry profiles at 6 and 12 hours post-induction. Lower right: proliferation of CLK1 RNAi line in mice. 1×105 trypanosomes were inoculated in 4 mice and RNAi was induced with doxycycline (Dox, as indicated) in 2 mice 48 hr later. Uninduced mice were culled as indicated () when their parasitaemias rose above 108 cells ml−1.