Text S1 - Iron Overload Favors the Elimination of Leishmania infantum from Mouse Tissues through Interaction with Reactive Oxygen and Nitrogen Species

Effect of iron overload on the carbonylation of proteins, peroxidation of lipids and integrity of DNA in the mouse liver. Figure S1. BALB/c mice were i.p. injected with saline solution or 10 mg of iron (-dextran, in a single dose) and were infected 15 days later by the i.v. route, with 2×107 L.infantum stationary promastigotes. Mice were sacrificed 60 days later and liver (100 mg) samples were collected and homogenised in protein lysis buffer. Liver protein lysates were reacted with DNP-hydrazone and then separated by SDS-PAGE followed by Western blotting (n = 4–5). No differences in the protein carbonyl levels were observed between control and iron-treated mice. Figure S2. C57BL/6 mice were fed a control (A) or a 2.5% iron-carbonyl diet (B) for 15 days. Liver samples were assayed by immunofluorescence to detect 4-hydroxynonenal (4-HNE) staining (green). Nuclei were counterstained with DAPI (blue). No 4-HNE staining was detected in the liver tissue of BALB/c mice treated for 15 days with saline solution or 10 mg of iron-dextran prior to a 30- and 60-day infection with L.infantum (staining was identical to A). Figure S3. BALB/c mice were i.p. injected with saline solution (A) or 10 mg of iron (B) (-dextran, in a single dose) and were infected 15 days later by the i.v. route, with 2×107 L.infantum stationary promastigotes. Mice were sacrificed 60 days later and liver samples were assayed by immunofluorescence to detect TUNEL staining (green). Nuclei were counterstained with DAPI (blue). No TUNEL staining was observed in animals receiving saline solution or iron treatment, except in the positive control (C, sample treated with DNase I). (DOCX)