Fig 2: Comparison of the evaluated activity of the statins used in this study against two strains of N. fowleri (ATCC 30808, ATCC 30215) and the induced cytotoxicity in the murine macrophage cell line (ATCC TIB-67); the reference drug amphotericin B was included as a positive control and to compare the obtained values.

Fig 3: POPC liposomes trigger an inflammatory response in macrophages. (a) Resident naïve peritoneal macrophages were obtained from peritoneal cavity lavage as described in the Section 2. Briefly, peritoneal cells were allowed to attach for 1 h at 37 °C in a CO2 incubator. Non-adherent cells were removed, and fresh medium was added. Cells were then incubated for an additional 16 h, and the presence of macrophage markers CD11b and F4/80 was evaluated by flow cytometry. (b) Bone marrow-derived macrophages (Mϕ) were obtained from the femur and tibia of male CD-1 mice (9 weeks old). After isolation, bone marrow cells were depleted of red blood cells and resuspended (2 × 106 cells/mL) in a culture medium supplemented with 10 ng/mL recombinant M-CSF. The culture medium was changed after 3 days, and non-adherent cells were discarded. The presence of mature bone marrow-derived Mϕ was evaluated by expression of CD11b and F4/80 and stimulated on day 7. (c) The macrophage cell line J774A.1 was obtained from ATCC and maintained in RPMI1640 with L-glutamine and penicillin/streptomycin and supplemented with 10% FBS. All macrophage types were exposed to POPC liposomes (2 × 105 liposomes per cell) for 1 h. Levels of TNF-α, IL-6, and IL-10 mRNA were determined by qPCR with the housekeeping gene GAPDH used to normalize data to cDNA inputs. Results are expressed as means ± SEM, and statistical analysis for the comparison between groups was performed using unpaired Student’s t-test with * indicating p < 0.05.

Fig 4: Dose–response curves of Nitroxoline against N. fowleri and murine macrophages J774A.1 (ATCC® TIB-67). The amoebicidal activity of Nitroxoline against ATCC® 30808™ (A) and ATCC® 30215™ (B) strains of N. fowleri after 48 h is shown. In addition, the cytotoxicity effect against murine macrophages (C) was measured after 24 h of incubation with the drug. Activity and cytotoxicity experiments were performed using three independent assays, as illustrated in the graphical legends.