RNA Seq of Leishmania infantum PP75 resistant to trivalent antimony
The resistant line (LiSbR) was previously selected in vitro by step-wise increase of SbIII drug pressure and the resistance index is 4-fold higher than of its susceptible counterpart (Liarte and Murta, 2010). These parasites were maintained in the culture under SbIII pressure and the new IC50 values for LiWTS and LiSbR were 0.12 mg/ml and 1 mg/ml, respectively, showing resistance index of 8-fold. The SbIII concentrations used in this study were obtained after detailed analysis of parasite viability. Different SbIII concentrations were used and the parasites were submitted to FACS analysis, using propidium iodide and annexin V (Immuno Chemistry Technologies). The antimony susceptible line (LiWTS) was cultivated during 24h in the absence of the drug (LiWTS 0 sample) or at a SbIII concentration of 0.06 mg/mL (LiWTS 0.06 sample) that corresponds to half of the SbIII IC50 for this LiWTS line, which does not induce apoptosis. For comparative analysis, the LiSbR line was also cultivated during 24h at the concentrations of 0.06 mg/mL (LiSbR 0.06 sample - same SbIII concentration of LiWTS line) Three independent biological replicates of each condition were cultured. Promastigote forms of these lines were grown at 26°C in M199 medium supplemented with 40 mM HEPES pH 7.4, 1 μg/mL biotin, 5 μg/mL hemin, 2 μg/mL biopterin, 2 mM L-glutamine, 500 U penicillin, 50 μg/mL streptomycin and 10% (v/v) heat-inactivated fetal calf serum (Liarte and Murta, 2010). Cells were washed in RPMI medium, pelleted by centrifugation and frozen at -70°C.
SUB2686584
SbR_0.06_C