FIG 4.

HSP83 RNAi causes severe growth defect and cytokinesis inhibition. (A) Growth of trypanosomes engineered to express, under tetracycline (Tet) control, stem-loop HSP83 RNA. Values are from 23 growth curves representing 9 clones. The clone used for follow-up work is represented by six independent inductions. Geometric mean ± SD; n = 3 to 21 at each point. (B) Northern blots of RNA from cells before and after induction of HSP83 RNAi, probed for HSP83 mRNA, and reprobed for tubulin mRNA and size markers; loaded with 700 ng RNA. Approximate lengths calculated from simultaneous size markers (not shown). Quantitation is given in Fig. S4A in the supplemental material. (C) Western blot of proteins from trypanosomes induced for RNAi of HSP83, probed with anti-human Hsp90 to reveal HSP83 (83 kDa) and an unknown cross-reacting band (50 kDa). Loaded by cell equivalents; quantitation given in Fig. S4B. (D) Nuclear and kinetoplast contents of trypanosome populations at increasing intervals after HSP83 RNAi induction. Inset, examples of abnormal 2N2K and 2N1K trypanosomes after 24 and 48 h of HSP83 RNAi, respectively (merged phase-contrast and DAPI fluorescent images). White bar, 1 μm. Data are means ± SD from independent experiments; n = 3 to 6 at each time point. **, P < 0.01; t test, two-tailed equal variance to 0-h control.