524 N. Sienkiewicz, S. Jarosławski, S. Wyllie and A. H. Fairlamb Fig. 5. Phenotypic analysis WT and DKO cells grown in the presence or absence of thymidine. Cells were grown in either the absence or presence of thymidine (dT) for 24 h and prepared for FACS, SEM, Giemsa, DAPI and TEM analysis. The histograms from the FACS analysis are presented in (A) WT minus thymidine, (B) DKO plus thymidine, (C) DKO minus thymidine with SEM, Giemsa and DAPI images of the respective cells in the right-hand panels. TEM showing the morphology of the WT (D), DKO plus thymidine (E) and DKO minus thymidine (F) are depicted. Bars represent 1 mm. N, nucleus; NM, nuclear membrane; FR, ﬂagella rod; FP, ﬂagella pocket; Ax, axonemes; G, glycosome; L, lysosome; PR, paraxial rod; MT, microtubules. normal WT and DKO sections (Fig. 5D and E respectively). The nucleus is also enlarged and is asso- ciated with a noticeable increase in the number of glyco- somes and lysosomes present throughout the sections compared with normal WT and DKO cells. No evidence for death by an apoptotic-like event was detected, either by staining live cells with Annexin V-FITC (Wyllie and Fairlamb, 2006) or by analysis of DNA for characteristic fragmentation patterns (Ioannou and Chen, 1996) (data not shown). Effect of antifolate and PTR1 inhibitors A range of DHFR–TS and PTR1 inhibitors were tested against WT and DKO trypanosomes to assess speciﬁcity against their predicted targets (see Fig. 1). As folate levels in the culture medium can also modulate drug sensitivity, EC50 values for growth inhibition were determined in high- (HMI9; 8 mM) and low- (FDM; ~30 nM) folate media (Table 1). In high-folate media, none of the nine inhibitors tested were particularly effective against WT cells (EC50 values > 1 mM), whereas in low-folate media WT cells became 68- to 310-fold more sensitive to the folate struc- tural analogues, methotrexate, aminopterin and raltitr- exed, with EC50 values ranging from 37 to 72 nM. The toxicity of the other inhibitors against WT cells was similar in both media. With DKO cells, none of the inhibitors showed any increase in sensitivity when cultured in low- folate media. Compared with WT, DKO cells are approxi- mately ﬁvefold less sensitive in high-folate medium to the DHFR inhibitors, aminopterin and methotrexate. Strik- ingly, the DKO parasites were ~1000-fold less sensitive © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533