Fig. 5

Effects of V-ATPase depletion on the uptake of lectin and dextran in BSF Trypanosoma brucei. a The BSF trypanosomes were allowed to endocytose DyLight 594-tomato lectin, at the indicated temperatures for 30 min, and were fixed and stained with DAPI. TbVAB depletion in BSF T. brucei was induced by withdrawing tetracycline for 24 h prior to the tomato lectin incubation. Scale-bar: 10 μm. b Control and TbVAB cKO 24 hpd cells were incubated with DyLight 594-tomato lectin at 37 °C for 30 min to allow lectin uptake. Values represent the mean fluorescence for 33 cells ± SD. The unpaired t-test indicated a significant difference (*P < 0.05) between control and TbVAB cKO 24 hpd cells. c Internalization of tomato lectin decreased after the induction of TbVAB knockout (cKO) and TbVAA knockdown (KD). Cells were incubated with DyLight 488-tomato lectin at 4 °C and then transferred to 37 °C for 16 min to activate endocytosis. Fluorescent intensities distributions of 30,000 cells were analyzed. AU, arbitrary units. d, e Flow cytometry analysis of tomato lectin and dextran uptake after TbVAB knockout and TbVAA knockdown. Cells were incubated with DyLight 488-tomato lectin (d) or dextran Alexa Fluor 488 (e) at 4 °C and subsequently transferred to 37 °C for the time indicated. Mean fluorescence intensity values of 30,000 cells were normalized against the 4 °C incubated cells. The unpaired t-test analysis of lectin uptake indicated a significant difference (*P < 0.05) between TbVAB cKO 0 hpd and 24 hpd cells