Fig 3.
TbRME-8 RNAi causes a severe proliferative defect, associated with a block in cytokinesis. (A) qRT-PCR of control and tetracycline-induced RNAi lines for TbRME-8 shows a 50% decrease in the RME-8 mRNA within 24 h of induction. arb., arbitrary. (B) Growth curves of control and tetracycline-induced RNAi lines for TbRME-8 show a severe growth defect within 24 h postinduction. Representative results for one of the two clonal cell lines studied are shown (all subsequent experiments were performed using this cell line). Cultures were diluted daily to maintain cell densities between 105 and 2 × 106 cells/ml, but the cumulative effect is shown. (C) Quantitative analysis of copy numbers of nuclei and kinetoplasts following RNAi induction for TbRME-8. A modest disruption in cell cycle progression (increase in 2K2N postmitotis cells) was observed within 24 h after RNAi induction (Day 1), and abnormal >2K2N cells accumulated within 48 h after RNAi induction (Day 2). The data and error bars are based on analysis of 100 cells each from two different clones. Examples of cells in each division state are shown: phase images merged with DAPI staining for DNA in the nucleus and the kinetoplast. The scale bar, bottom right, is 2 μm.