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Fig. 4. Polymorphic phenotype of the TbSPT2 RNAi line. (A) Scanning electron micrographs of TbSPT2 RNAi cells grown for either 3 or 6 days in the presence of Tet. Compared with 29-13 (WT) cells, many TbSPT2 RNAi +Tet cells showed incomplete cytokinesis (Days 3a and 3b). In some TbSPT2 RNAi +Tet cells, the parental and daughter flagella both emerged from the same flagellar pocket, with the daughter flagellum partially detached from the parent flagellum and the cell body (Day 3c). At day 6 post Tet induction, many enlarged TbSPT2 RNAi cells containing polyflagellated extensions were observed (Days 6a and b). (B) DNA content analysis of the TbSPT2 RNAi line with (+) or without (–) Tet over 5 days of treatment. The x-axis shows the DNA content, measured by intensity of fluorescence emission at 440 nm. The y-axis shows the number of cells. The number of cells with a DNA content of 4C increased on days 1-2; number of cells with a DNA content of >4C and <2C increased progressively on days 3-5. The same was observed upon treating the cells with increasing concentrations of myriocin for 24 hours. (C) TbSPT2 RNAi –Tet and +Tet cells at day 3, and 29-13 cells treated with 1.5 µM myriocin for 24 hours were analyzed by immunofluorescence microscopy by using antiserum specific for the paraflagellar rod (ROD1). Cells were also visualized by phase microscopy and by using DAPI staining. (i) control TbSPT2 RNAi–Tet. (ii-v) TbSPT2 RNAi + Tet. (vi-vii) 29-13 cells treated with 1.5 µM myriocin. Both treatment conditions produced similar phenotypes, with TbSPT2 RNAi +Tet cell images being representative of myriocin-treated cells and vice versa. (ii) A cell stuck partway through cytokinesis, undergoing another round of DNA replication. Note that the kinetoplast has already been re-segregated in the upper part of the cell. (iii) Double (0N2K) zoids (left) and single (0N1K) zoids (right) – anucleate cells with two or one kinetoplasts, respectively; the 0N2K-zoid has a partially detached daughter flagellum. (iv) 2N1K* cell (containing an undivided kinetoplast) with a detached daughter flagellum. (v) A 2N1K cell (left) and a 4K2N cell with a detached flagellum (right). (vi) A 2N2K cell with a mispositioned cleavage furrow that can give rise to a zoid and a 2N1K cell. (vii) A 4N3K cell with one undivided kinetoplast, one detached flagellum, and a cytoplasmic tether. Arrows indicate cleavage furrows and cytoplasmic tethers.
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