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. 2016 Mar 3;10(3):e0004308. doi: 10.1371/journal.pntd.0004308

Fig 4. Assay of enzyme activity, argininosuccninate production, western blot analysis, assay of reduced thiol pool and polyamine level.

Fig 4

A) ASS enzyme activity of WT L. donovani cell line under stress conditions. Exposing the WT cell line to the stress inducing reagents, menadione (oxidative stress) or a mixture of menadione and SNAP induced a ~1.8-and ~1.5-fold increase in enzymatic activity, respectively was observed. About 100% enzymatic activities were blocked when WT cells were incubated with 5 mM α-methyl-DL-aspartic acid (MDLA), (a specific inhibitor of ASS). B) Bar-graph showing the amount of ASA produced in different in WT L. donovani exposed to different stress inducing reagents. The argininosuccinate levels were increased ~1.5-fold after exposure to different stress inducing reagents in comparison to WT cell line, whereas the level of argininosuccinate was decreased to the level of blank in presence of inhibitor (5 mM MDLA). C) Western Blot image of LdASS of wild type and OE cell lines. Left panel-under different stress conditions i.e. menadione (M), SNAP (S), combination of SNAP and menadione (S+M). Right panel (lanes 5–6) L-arginine limiting conditions after L. donovani cells were cultured in L-arginine free medium for two successive generations. D) Densitometry analysis of the Western Blot results. E) Bar-graph showing the amount of reduced thiol pool in WT, OE and LdASS-/-/+ cell lines under different stress conditions and in presence of two different inhibitors of enzymes involved in polyamine biosynthesis pathway. The reduced thiol level was found to be decreased in LdASS-/-/+ cells by ~3-fold compared to WT and OE cell lines, which were further decreased when exposed to different type of stress. DFMO and MTPA were used as inhibitor of ornthine decarboxylase (ODC) and spermidine synthase (SPS) respectively. Reduced thiol level was found to be decreased in LdASS-/-/+ cell line and under stress conditions. F) Bar-graph showing the relative level of two polyamines i.e. putrescine and spermidine in different L. donovani cell lines (WT, VC, OE and LdASS-/-/+). The level of both the polyamines in the LdASS-/-/+ was found to be decreased by ~4 fold compared to WT parasites whereas ~2.4 fold increased level was observed in OE parasites compared to WT parasites. Each measurement was carried out in triplicate and data are expressed as the mean. Vertical lines on top of the bars represent s.e. values and star symbol above the vertical lines denotes significant changes with P<0.005.