Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability
Figure 5
CsA-treated L. donovani promastigotes show altered morphology.
Promastigotes were incubated with 0.15% ethanol or 15 µM (B, C) or 20 µM (A) CsA at 26°C, pH 7.4 for 72 hours. Axenic amastigotes were prepared as described in experimental procedure. 107 cells were fixed with either methanol for Giemsa staining (A), or 2.5% glutaraldehyde for scanning electron microscopy (B). The bar corresponds to 1 µm (B) and 5 µm (A). Two independent experiments were performed and representative fields are shown. (C) Flagellum length measurement. CsA-treated and solvent treated cells L. donovani promastigotes were fixed in methanol and stained with anti-tubulin monoclonal antibody. Flagellum length was measured from a total of 180 cells each for control and CsA-treated samples. Only cells with a single flagellum that was completely visible and fully in focus were taken into account. Samples were observed with a DMR Leica microscope and images were captured with a Cool Snap HQ camera (Roper Scientific). Images were analysed using the IPLab Spectrum 3.9 software (Scanalytics & BD Biosciences) and flagellum length was measured using ImageJ (NIH). (D) Immunoblot analysis of CsA treated parasites. Parasites were treated with solvent or 15 µM CsA for 72 hours, lysed in 1× Laemmli buffer, and lysates equal to 2×107 cells were analyzed by immunoblotting. Promastigote specific marker LPG (upper), amastigote specific marker A2 (middle) and α-tubulin (lower) were analyzed. Two independent experiments which gave identical results were performed.