Figure 1

RNAi of CRK9. Procyclic form (29-13) cells were transfected with a pZJM vector carrying a short (300 bp) fragment of CRK9. Transfected cell lines were selected using phleomycin and cloned. (A) RNAi was induced by addition of tetracycline (1.0 μg/ml) and cell growth monitored daily using a hemocytometer. Daily dilution of cell cultures with fresh medium was performed when the cells grew at a linear rate. Insets show mRNA levels of CRK9 and tubulin determined by real time quantitative RT-PCR; levels are shown relative to those of un-induced cells. (B) Cells were harvested daily, fixed and stained with PI, followed by DNA content analysis using flow cytometry. The proportion of cells in each phase of the cell cycle was determined using the ModFit software and the proportions of G1, S and G2/M phase cells were plotted each day. (C) Cell sorting histograms of cell distribution by DNA contents. The small peaks next to the y-axis represent cell debris. (D) The PI-stained cells were also examined visually using fluorescence microscopy and the numbers of 1N1K, 1N2K and 2N2K cells determined as percentages of a total population of ~200 cells after 5 days of CRK9 RNAi (N, nucleus; K, kinetoplast).