Figure 1.

Estimation of S-phase duration in T. brucei TREU927. (A,B) The doubling time (dt) for procyclic forms of T. brucei was estimated to be 8.5 h (r2 = 0.9906). This estimate was confirmed taking the values at exponential phase and using Doubling Time software (http://www.doubling-time.com). Error bars indicate SD of three independent experiments. (C) DAPI-labeled parasites (2K2N) were used to measure the percentage of parasites in cytokinesis, which was estimated to be 6.99 ± 1.13% (n = 358). Error bars represent SD. The scale bars for the fluorescence images correspond to 2 µm. This value was used in Eq. 1 (19) to estimate the cytokinesis-phase duration. (D) To estimate the duration of the G2 + M phases, EdU was added to the culture, and parasites were continuously collected every 15 min until parasites containing two EdU-labeled nuclei in the same cell (cytokinesis) were observed. This pattern was observed after 2 h. This assay was carried out in triplicate, and in all replicates, we found a parasite containing two EdU-labeled nuclei at the same time. Scale bar = 2 µm. (E) EdU-labeled parasites (after 1 h EdU pulse) were used to estimate the percentage of parasites able to uptake this thymidine analog (39 ± 2.7%). Error bars represent SD. Scale bar = 2 µm. This value was used in Eq. 2 to estimate the S-phase duration. These assays were carried out in biological triplicate (n = 358 parasites). (F) New estimates for G1- and S-phase duration. ccu means cell cycle unit, where one unit corresponds to the specific doubling time for each strain.