Figure 5

Expression of sense and antisense Ldccys2 transcripts in L. (L.) chagasi and survival of wild type, sense and antisense Ldccys2 expressing amastigotes in macrophages. (A) Northern blot analyses of amastigotes expressing sense and antisense transcripts. Total RNA (10 μg/lane) isolated from axenic amastigotes of wildtype parasites, transfectants with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid were separated on 1.2% (w/v) formaldehyde agarose gel and blotted on to Hybond N+ membrane. Blots were probed with probes specific for antisense (panel 1) and sense (panel 2) transcripts. Panel 3 represents ethidium bromide stained gel. (B) Western blot analysis of sense and antisense Ldccys2 expressing amastigotes. Equal amounts of total protein extracted from axenically transformed amastigotes with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid and wildtype parasites were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond P membrane. The blot was probed with α-Ldccys2 antibody (panel 1) and a duplicate gel was stained with Coomassie blue (panel2). (C) Bar graph showing total number of amastigotes within the macrophage cells. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amstigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining.(D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.