Fig. 7. Effect of triple knockdowns of CRK1, CRK2 and CycE1/CYC2 on the cell cycle progression and morphology of bloodstream-form cells. (A) Cloned cells harboring the CycE1/CYC2+CRK1+CRK2 RNAi plasmid construct were incubated at 37°C without (-Tet) or with 1.0 µg/ml tetracycline (+Tet). Cell growth was monitored daily and cell numbers plotted on a logarithmic scale. The inset shows the semi-quantitative RT-PCR estimation of intracellular mRNA 3 days after RNAi induction with -tubulin mRNA (TUB) included as a sampling control. (B) Cells sampled on day 1 to 4 were stained with propidium iodide and subjected to FACS analysis for DNA content. The histograms from the FACScan are presented on the left, and the percentages of cells in G1, S and G2-M phases determined by the ModFitLT software are plotted on the right. (C) Cells with different numbers of nuclei (N) and kinetoplasts (K). Data are presented as the mean percentage (±s.e.) of total cells counted (>200) from three independent experiments. (D) BrdU incorporation into cells 3-5 days after RNAi induction. Arrows indicate where BrdU is not incorporated into the nuclei. (E) A triple knockdown cell stained with YL1/2 antibody for newly synthesized microtubules: none was found at the posterior end.