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. 2011 Aug;10(8):1082–1094. doi: 10.1128/EC.05098-11

Fig. 7.

Fig. 7.

TbAZI1 knockdown produces a block in cytokinesis. (A) Growth curves of p2T7-177-TbAZI1 PTT cells. Cell cultures were grown in triplicate in the presence (open squares) or absence (filled squares) of tetracycline (1 μg ml−1) and maintained under mid-logarithmic growth conditions by periodic dilution (indicated by breaks in the growth curves). Cell density was assessed by using a Coulter Counter. Data points are mean values ± standard error. TbAZI1 knockdown by RNAi was confirmed by real-time RT-PCR. The histogram shows the loss of TbAZI1 transcript 24 h after induction. (B) Growth curves of p2T7TA-TbAZI1 SMB cells. Cell cultures were grown in triplicate in the presence (open squares) or absence (filled squares) of tetracycline (1 μg ml−1). Cell density was assessed by using a Coulter Counter. Data points are mean values ± standard error. TbAZI1 knockdown by RNAi was confirmed by real-time RT-PCR. The histogram shows the loss of TbAZI1 transcript 24 h after induction. (C) Histogram representing cell cycle analysis of TbAZI1 RNAi cells and the accumulation of abnormal cellular forms. Uninduced and induced cells were fixed and mounted with Vectashield containing DAPI to stain for DNA. Numbers of nuclei and kinetoplasts per cell were counted at 24, 48, and 72 h; at least 200 cells were counted for each time point. (D) TbAZI1 knockdown cells manifest a delay in cytokinesis. RNAi cells were induced for 24 h, and then uninduced and induced cells were fixed and DAPI stained. Mitotic cells at different stages of cytokinesis were assessed by light microscopy. Numbers represent cells that have replicated their DNA but not initiated cytokinesis (upper panels), cells that have started ingression of the cleavage furrow (middle panels), and cells that have not yet undergone abscission (bottom panels). Two hundred cells were counted. (E) Distance between kinetoplasts was measured in at least 30 2K2N cells by using Metamorph (P < 0.05). Filled bars represent uninduced cultures, and open bars represent induced cell cultures. Bars, 2 μm.