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Fig. 6. TbSPT2 RNAi growth defect and cytokinesis defect can be rescued with 3-KDS, but not ceramide. (A) TbSPT2 RNAi cell lines grown in the presence of Tet were supplemented with either 1 µM 3-KDS or 5 µM C-18 ceramide (Cer) and compared with uninduced (–Tet) RNAi cells. Cultures were initiated in SDM79 medium at a density of 10⁶ cells/ml and culture densities were determined daily for 6 days using a Coulter counter. TbSPT2 RNAi +Tet cells complemented with 3-KDS grow to ten times the number of those without 3-KDS. Ceramide complementation has no effect on cell number. (B) Flow cytometric analysis of DNA content of the TbSPT2 RNAi cell line complemented with 3-KDS and ceramide as described above, shown 3 days post Tet induction. To assess DNA content, timed samples were collected and stained with 20 µg/ml Hoechst dye solution containing 0.1% Triton X-100 and 0.5% formaldehyde, and analyzed using BD LSRII Flow Cytometer. Cell numbers are given at the x-axis, DNA content as measured by intensity of emission at 440 nm is given at the y-axis. A clear decrease in 2C DNA content and an increase in >4C and <2C DNA content is seen with addition of 3-KDS but not ceramide to TbSPT2 RNAi cell line induced with Tet.

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