Characterization of L. (L.) chagasi heterozygous knockout mutant amastigotes and intra macrophage survival of Ldccys2KO amastigotes in vitro . (A) Northern blot analysis. Total RNA (10 μg/lane) from U937 cells infected with wild type promastigotes, Ldccys2KO and uninfected U937 cells were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with Ldccys2 coding region DNA probe (1) and α-tubulin gene from L. (L.) chagasi (2). (B) Western blot analysis. Equal amount of lysates from U937 cells infected with wild type promastigotes, Ldccys2KO promastigotes and uninfected U937 cells were separated on 10% (w/v) SDS-PAGE, blotted on to Hybond P membrane. Panel 1 represents the membrane that was probed with α-Ldccys2 antibody and panel 2 is the duplicate gel stained with Coomassie blue. (C) Bar graph showing the number of intracellular amastigotes. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amastigotes 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.