FIG 3.

(A) Growth curves of T. brucei BF DUT-KO and BF parasites in the presence of dThd or 5-Met-dCtd. (B) Growth curves of T. brucei BF DUT-KO in the presence and absence of dThd and of T. brucei BF DUT-KO cells expressing human dCMP deaminase (DCTD). Each time point corresponds to the mean for triplicate biological replicates. (C and D) Western blot analyses showing that the T. brucei BF DUT-KO cell line lacks dUTPase and that human DCTD is successfully expressed in doxycycline-induced cells. Anti-TbdUTPase (1:75,000), anti-DCTD (1:1,000), and anti-β-tubulin (1:5,000) antibodies were used. Each lane contained 5 × 10⁶ parasites. (E) DCTD subcellular localization in T. brucei BFs expressing human DCTD analyzed by immunofluorescence. Anti-DCTD and Alexa Fluor 488-conjugated goat anti-mouse antibodies were used. Mitochondria were stained with MitoTracker Red CMXRos, and nuclei and kinetoplasts were stained with DAPI. Images were captured using an Olympus IX81 microscope and deconvolved with Huygens Essential software (version 3.3; Scientific Volume Imaging). Images were analyzed by using Fiji software. Bar, 5 μm.