Fig 1: NDKb and EF2 expression levels in wild-type (WTS), SbIII-resistant (SbR) and clonal lines from L. braziliensis and L. infantum untransfected or transfected with the constructs pIR1BSD (empty vector), pIR1BSD-NDKb or pIR1BSD-EF2. Total proteins (20 µg) were separated by electrophoresis on 12% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The profiles of total proteins stained with Coomassie blue are shown. The blots were probed with rabbit polyclonal anti-NDKb (1:200) (a, b and c) or rabbit monoclonal anti-EF2 (1:200) (a, d and e) antibodies and developed using ECL Plus kit. All membranes were incubated with the anti-a-tubulin monoclonal (1:15,000) antibody for normalization of the results. Quantification of the bands was done by densitometric analysis using the software GelAnalyzer 2010. The figure is representative of all results obtained from two different biological replicates of each sample. The values of each replicate were used to calculate the averages and for determination of the ratios presented for each protein analyzed
Fig 2: EC50 of lamivudine for wild-type and NDKb-overexpressing L. braziliensis lines (a) and effect of lamivudine on the growth of L. braziliensis lines upon SbIII exposure (b). Parasites were incubated in M199 medium in the absence or presence of different concentrations of lamivudine (C8H11N3O3S) (125 to 10,000 µM). For competition assay, cells were exposed to the EC50 of SbIII (7, 12 and 15 µM for the LbWTS and NDKb-overexpressing clones 4 and 9, respectively) and the EC50 of lamivudine (766, 2110 and 2014 µM for the LbWTS and NDKb-overexpressing clones 4 and 9, respectively) independently or combined, followed by incubation for 48 h. The percentage of relative growth was determined using a Z1 Coulter Counter. Mean values ± standard deviations from three independent experiments in triplicate are shown. Statistical analysis was carried out using Student’s t-test. Statistically different values are highlighted as follows: **P < 0.01; ***P < 0.001. Pairwise comparisons (a): 250 µM: LbWTS vs LbNDKb clone 9 (t(7) = 4.04, P = 0.0049); 500 µM: LbWTS vs LbNDKb clone 4 (t(9) = 5.27, P = 0.0005); LbWTS vs LbNDKb clone 9 (t(9) = 3.74, P = 0.0046); 1000 µM: LbWTS vs LbNDKb clone 4 (t(10) = 5.67, P = 0.0002); LbWTS vs LbNDKb clone 9 (t(10) = 3.52, P = 0.0055); 2500 µM: LbWTS vs LbNDKb clone 4 (t(8) = 5.03, P = 0.0010); LbWTS vs LbNDKb clone 9 (t(8) = 3.91, P = 0.0045)
Fig 3: SbIII susceptibility assay of clonal lines from L. braziliensis (a) and L. infantum (b) non-transfected or transfected with the constructs pIR1BSD or pIR1BSD-NDKb. Parasites were incubated in M199 medium in the absence or presence of different concentrations of SbIII (1.17 to 599.04 µM) for 48 h and the percentage of relative growth was determined using a Z1 Coulter Counter. Mean values ± standard deviations from three independent experiments in triplicate are shown. Statistical analysis was carried out using Student’s t-test. Statistically different values are highlighted as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Pairwise comparisons (a): 1.17 µM: LbWTS vs LbNDKb clone 4 (t (5) = 9.47, P = 0.0002); LbWTS vs LbNDKb clone 9 (t (9) = 3.36, P = 0.0084); 2.34 µM: LbWTS vs LbNDKb clone 4 (t (5) = 4.3, P = 0.0077); LbWTS vs LbNDKb clone 9 (t (8) = 3.74, P = 0.0057); 4.68 µM: LbWTS vs LbNDKb clone 4 (t (4) = 4.02, P = 0.0159); LbWTS vs LbNDKb clone 9 (t (5) = 4.99, P = 0.0042); 9.36 µM: LbWTS vs LbNDKb clone 4 (t (6) = 8.39, P = 0.0002); LbWTS vs LbNDKb clone 9 (t (8) = 12.50, P < 0.0001); 18.72 µM: LbWTS vs LbNDKb clone 4 (t (8) = 2.88, P = 0.0206); LbWTS vs LbNDKb clone 9 (t (10) = 7.36, P < 0.0001)
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