Fig. 4

Effect of V-ATPase depletion on the uptake of transferrin in the bloodstream forms of Trypanosoma brucei. a Trypanosomes were allowed to endocytose transferrin Alexa Fluo 488, at the indicated temperatures for 16 min, and were fixed and stained with DAPI. TbVAB depletion in BSF T. brucei was induced by withdrawing tetracycline for 24 h before the transferrin incubation. Scale-bar: 10 μm. b Control and TbVAB cKO 24 hpd cells were incubated with transferrin Alexa Fluo 488 at 37 °C for 16 min to allow transferrin uptake. Values represent the mean fluorescence for 72 cells ± SD from three technical replicates (n = 1). The unpaired t-test indicated a significant difference (*P < 0.05) between control and TbVAB cKO 24 hpd cells. c Internalization of transferrin increased after the induction of TbVAB knockout. Cells were incubated with transferrin Alexa Fluo 488 at 4 °C and then transferred to 37 °C for 16 min to activate endocytosis. Fluorescent intensities distributions of 30,000 cells were analyzed and shown in the flow cytometry diagrams. AU, arbitrary units. d Flow cytometry analysis of transferrin uptake after TbVAB knockout and TbVAA knockdown. Cells were incubated with transferrin Alexa Fluo 488 at 4 °C and subsequently transferred to 37 °C at the times indicated. Mean fluorescence intensity values of 30,000 cells were normalized against the 4 °C incubated cells. Unpaired t-test analysis indicated a significant difference (**P < 0.01) between TbVAB cKO 0 hpd and 24 hpd cells