Processing of GFP-TbAtg8 during parasite growth in culture.

<p><i>T. brucei</i> procyclic forms expressing GFP-TbAtg8.1 were cultured in mid-log phase for several days, then diluted to 3×10⁵ cells/mL at day 0 and kept in culture without further dilution for 7 days. (A) Parasite density (mean values ± standard deviations from three independent cultures). (B) Each day, GFP-TbAtg8.1 fluorescence was determined by flow cytometry before (•) and after (▪) starvation for 2 hours in gHBSS and expressed relative to the values obtained at day 1 (upper panel). The differences between GFP-TbAtg8.1 fluorescence before and after starvation were plotted to obtain a number for the capacity of parasites to undergo autophagy. The values are expressed relative to the difference observed on day 1. (C) Immunoblot of parasite lysates (5×10⁶ cell equivalents per lane) before (−) and after (+) starvation probed with antibodies against GFP (lower panel) or eukaryotic elongation factor 1A (upper panel, used as loading control). (D) Quantification by fluorescence microscopy of GFP-TbAtg8.1 puncta per cell and cells containing puncta in parasites before (•) and after (▪) starvation. At each time point, at least 100 cells per experimental condition were counted. Error bars indicate standard deviations from three independent experiments.