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Figure 5


Fig. 5. Phenotypes induced by RNAi in procyclic cells. (A) A small population of cells (~1%) displayed flagella pairs. Cytoskeletons of cells induced for 48 hours probed with YL1/2 (b) and M25 plus DAPI (c). (B) A small population of cells displayed clusters of non-flagellated basal bodies. Cells were probed as in A. (C) A RNAi-induced four-basal-body cell, probed with YL1/2 (b) and M25 plus DAPI (c), illustrating a normal phenotype. Knockdown of TbNRKC does not block basal body separation or cytokinesis. Bars, 5 µm. (D) Basal body counts of wild-type (WT) and TbNRKC RNAi-induced cells (+TET). After 72 hours of induction, 46.2% of the population had two basal bodies (2BB) compared with 77.04% of wild-type cells. 47.8% of induced cells had four basal bodies (4BB) (two mature and two immature) compared with 22.2% in wild-type cells. 4.6% displayed more than four basal bodies (>4BB), 0.4% displayed clustered basal bodies (Clust. BB) and 1% displayed mature flagella pairs (MAT. PAIRS). Values are mean ± s.e.m. of three experiments. (E) FACS analysis of RNAi knockdown cells. After 72 hours (72 H) of RNAi induction, the percentage of cells in G1 decreased from 61.17% (WT cells) to 51.83%. The percentage of cells in S-phase remained stable (11.23% WT, 12.83% RNAi). The percentage of cells in G2-M phase increased (from 24.93% WT to 28.78% RNAi). The percentage of cells with abnormally high DNA content also increased (from 3.02% WT to 6.87% RNAi).





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