Figure 6.

Inhibition of LdTOPIA by norclomipramine leads to parasite elimination.A, DNA relaxation assay was carried out using (−) SC pBluescript DNA, purified (left panel) LdTOPIA (10 nM) and (right panel) EcTOPIA (10 nM) and in absence or presence of increasing concentration of norclomipramine. B, modified MTT assay using increasing concentration of norclomipramine or imipramine treated L. donovani DD8 parasites promastigotes and axenic amastigotes to monitor percentage of parasite killing. IC50 values were calculated from the graph plotted, percentage of parasites killed versus NCL/Imi concentration. (n = 3 and 3 biological replicates, mean ± SD). C, immunofluorescence analysis of 10 μM NCL-treated L. donovani DD8 to analyze R-loop formation. Parasites upon treatment for indicated timepoints were fixed and incubated with anti-LdTOPIA-AlexaFluor488, anti-DNA-RNA hybrid (S9.6)-AlexaFluor568 antibodies, and counterstained with DRAQ5. Scale Bar, 5 μm. D, graphical representation of the extent of R-loop formation estimated from the fluorescence intensity. [n = 60 (20 nuclei of 3 biological replicates), mean ± SD. p versus 0h]. E, DRIB assay was carried out from untreated and NCL treated, genomic DNA isolated and RNaseH treated samples and (F) densitometry of the same samples (n = 3 and 3 biological replicates, mean ± SD). G, flow cytometry of Raw264.7 infected with DD8-GFP treated with 10 μM norclomipramine. Graph showing the percentage of infected macrophages at each time point. H, microscopic images of the same. Scale Bar, 20 μm. I, intracellular parasite burden, 48 h treatment post-infection with DD8, BHU575 and BHU814. (n = 100 cell nuclei, values averaged and plotted for 3 biological replicates, mean ± SD).