Fig. 4. Generation of zoids through depletion of TbCentrin4, but not TbCentrin2. (A,B) Cells were induced for TbCentrin2-RNAi (A) or TbCentrin4-RNAi (B), and the DNA content of fixed cells was monitored using DAPI staining. Cells were divided into six categories: one kinetoplast and no nucleus (1K0N; zoids), one kinetoplast and one nucleus (1K1N), two kinetoplasts and one nucleus (2K1N), two kinetoplasts and two nuclei (2K2N), one kinetoplast and two nuclei (1K2N), and cells with multiple nuclei (Multinucleated). Results from two independent experiments were averaged for n=500 cells/time point. The number of zoids (1K0N) increased in cells depleted of TbCentrin4 but not of TbCentrin2. (C,D) Cells analyzed by flow cytometry using forward scatter to measure the cell size. 100,000 events were analyzed for each time point. (E,F) TbCentrin2-depletion led to larger cells, whereas depletion of TbCentrin4 generated two populations, the smaller cells mostly zoids (E), the larger cells mostly multinucleated (F). Scale bar, 5 µm.