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Fig. 1. RNAi silencing of TLK1 and TLK2 in the procyclic form of T. brucei. (A) Clonal cell lines harboring the respective TLK1 and TLK2 RNAi constructs were cultivated with (+Tet) or without (–Tet) tetracycline and monitored for cell growth. The insets show the levels of mRNA, monitored by northern blot, in cells before (–) and after (+) RNAi. -Tubulin (Tub) was included as a loading control. (B) Flow cytometry analysis of DNA contents in TLK1 or TLK2 RNAi cells. Insets show the percentages of cells at G₁-, S- or G₂/M-phases. (C) The control and TLK1 RNAi cells after 16 and 24 hours RNAi induction were tabulated for different numbers of nuclei (N) and kinetoplasts (K). The data are presented as the mean percentage±s.e. of 200 cells counted from three independent experiments. (D) Control and TLK1 RNAi cells after 24 hours induction were stained with L1C6 antibody to monitor the nucleolus, and with L8C4 and YL1/2 antibodies to label the flagella and basal bodies, respectively, and counterstained with DAPI to show the nucleus and kinetoplast. Arrows point to the basal bodies, and arrowheads indicate the flagella. (E) Spindle structures in the control and TLK1 RNAi cells after 24 hours of induction. Cells were stained with the KMX-1 antibody for the spindle and DAPI for DNA. Percentages of cells with the spindles were determined among 200 1N2K and 2N2K cells in three separate experiments. Bars, 2 µm.

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