Figure 9.

Effect of inhibition of l-arginine availability on regulation of polyamine production in Leishmania donovani-infected macrophages. THP-1 monocyte-derived MΦs (1 × 10⁶ cells/ml) were grown in l-arginine-supplemented and l-arginine-depleted RPMI medium followed by infection with L. donovani (multiplicity of infection 1:10) for 48 h (A,B). In a seperate experiment, THP-1 MΦs (1 × 10⁶ cells/ml) were either left untreated or preincubated with 250 µM NEM for 10 min followed by infection with L. donovani for 48 h (C,D). In another experiment, THP-1 MΦs (1 × 10⁶ cells/ml) were transfected with control siRNA, CAT-1 or CAT-2 siRNA followed by infection with L. donovani for 48 h (E,F). The polyamines (putrescine and spermidine) were extracted by TCA precipitation followed by dansyl chloride derivatization, separation by reverse-phase high performance liquid chromatography as described in “Materials and Methods.” Dansylated putrescine (A,C,E) and dansylated spermidine (B,D,F) were quantified by fluorescence spectrometry. Each experiment was repeated three times. Each determination was made in triplicate and the values were expressed as mean ± SD for three independent experiments. Kruskal–Wallis with Dunn’s multiple comparison test was used to evaluate statistical significance for comparing three or more groups; *p < 0.05; **p < 0.005; ns, non-significant.