TbKINX1B is essential in bloodstream forms. A. Left panel, western blot of BSF whole cell protein extracts from TbKINX1B^RNAi cell line in the absence (−) or presence (+) of tetracycline at 24 h, 48 h and 72 h using anti-TbKINX1B, and anti-tubulin (TAT1) as a loading control. Right panel, down-regulation of TbKINX1B in BSF (+) inhibits cell growth after 48 h of induction, as compared to non-induced cells (−) (n = 3). B. Left panel, IFA micrographs of WT and of 48 h induced TbKINX1B^RNAi BSF cell lines. Immunofluorescence shows that down-regulation of TbKINX1B leads to multi-flagellated and multinucleated cells. Flagella were labelled with anti-PFR2 (green), and kinetoplasts and nuclei were DAPI-stained (blue). A large FP or vacuole is marked with an asterisk. Scale bars 5 μm. Right panel, quantification of cell division phenotypes in WT and in TbKINX1B^RNAi cell lines after 24 h, 48 h and 72 h of induction. Normal phenotypes are underlined (200 cells, n = 3). C. Transmission Electron Microscopy (TEM) thin-sections of WT BSF and of TbKINX1B^RNAi induced for 48 h. The structural organization of WT cells reveal well-defined FP (black asterisk), FPC, Golgi (G), endoplasmic reticulum (ER), recycling endosomes (RE), glycosomes (GL), Flagellum (Fg), internal vesicles and kinetoplast (K) (a, b). TbKINX1B knock-down cells possess enlarged FP (black asterisk, c, d), with the disturbed endo-membrane organization (i.e., FP harboring dense material or enlarged) (e, f).