TbRBX1 depletion influences kDNA replication in the procyclic form of T.brucei.

(A) Growth curves of PCF (left panel) and BSF (right panel) parasites after tetracycline-induced TbRBX1 RNAi. PCF and BSF parasites transfected with pZJM-TbRBX1-RNAi construct were cultured in the absence (-TET) or presence (+TET) of tetracycline (1μg/ml). Insets in the respective growth curves show northern blot (PCF) or RT-qPCR (BSF) experiments demonstrating the RNAi-mediated downregulation of TbRBX1 mRNA. Error bars represent the ± SEM of 3 biological replicates. *: p≤ 0.01; **: p≤ 0.001. (B) Flow cytometry analysis of TbRBX1-RNAi PCF parasites. The x-axis shows fluorescence intensity in the FL2-A channel. The bottom panel indicates percentage of cells in different cell cycle stages as determined using the FlowJo 7.5.5 software and the Dean-Jett-Fox model. (C) Nuclei (N) and kinetoplasts (K) configurations of TbRBX1-RNAi PCF parasites induced (+TET) with tetracycline for the indicated times. At least 200 cells were counted for each time point from 3 biological replicates. (D) Images of DAPI-stained cells viewed by phase-contrast and fluorescence microscopy. The effect of depleting TbRBX1 on cellular morphology, the nucleus (N) and kinetoplast (K) are shown.