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. 2017 Mar 15;130(6):1179–1193. doi: 10.1242/jcs.194308

Fig. 5.

Fig. 5.

FAZ10 knockdown in BSF T. brucei impairs cell growth and causes flagellum detachment and cytokinesis defects. (A) Western blotting of 1×106 cells/lane with anti-Myc antibody of whole cell lysates of uninduced (−tet) and tetracycline-induced (+tet) FAZ10::Myc or FAZ10 RNAi+FAZ10::Myc cells. Anti-OPB antibody was used as a loading control. (B) Immunofluorescence of uninduced (−tet) and induced (+tet 48 h) FAZ10 RNAi+FAZ10::Myc cells. DIC, DAPI (blue), anti-Myc (FAZ10:myc, green) images are shown, as indicated. Arrowhead, detached flagellum. (C) Cumulative growth curve of FAZ10 RNAi cells, seeded at 1×105 cells ml−1 in the presence (+tet) or absence (−tet) of tetracycline. (D) Fluorescence microscopy of DAPI-stained nuclei and kinetoplasts of FAZ10 RNAi cells at different timepoints (images i–iii and vi are 24 h; images iv and v are 16 h) after induction with tetracycline. Arrowheads, partially or fully detached flagella. (E) Quantification of FAZ10 RNAi cells with flagellum detachment defects over time following RNAi induction. n>500 cells/timepoint. (F,G) Quantification of nuclei and kinetoplasts in DAPI-stained FAZ10 RNAi over time post-RNAi induction (n>150 cells/timepoint). (H) Fluorescence microscopy of a DAPI-stained FAZ10 RNAi 1N2K and 2N2K cells undergoing unequal cytokinesis 8 h after tetracycline induction. Arrows, cleavage furrows. Error bars show s.d. of three independent experiments. Scale bars: 5 µm.