Rpmi 1640 medium

All human cell lines were purchased from ATCC (LGC Standards, Molsheim, France). Human mammary (MCF-7, ATCC^® HTB-22) and colon LoVo adenocarcinoma (ATCC^® CCL-229^™) cell lines were maintained in DMEM high glucose medium (Dominique Dutscher, 67,172 Brumath, France, Cat No. L0102-500), while human acute monocytic leukemia cell line (THP-1, ATCC^® TIB-202) was maintained in RPMI-1640 Medium (ATCC^® 30-2001™, LGC Standards, Molsheim, France), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Life Technologies, Paisley, UK, Cat No. 10270-106) and 1% (v/v) penicillin–streptomycin (10,000 units/mL and 10,000 µg/mL, Life Technologies, Paisley, UK, Cat No. 15140-122). Cells were kept at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂ during their exponential growing phase and in the course of incubation with investigated compounds. Before confluence, adherent cells were trypsinized and subcultured twice a week.

The splenic tissues of three animals from groups B1, B2 and B3 (n = 9), which did not undergo tumor induction, were collected. Splenic tissues were compressed using the rough surface of two frosted microscope slides and the splenic homogenate obtained was washed with RPMI 1640 medium (LGC Biotecnologia, Cotia, Brazil) and resuspended with syringes and needles of decreasing size (18 G, 22 G and 26 G, respectively), until a homogeneous solution was obtained. Splenic homogenates were centrifuged at 200 g for 10 min and resuspended in 360 μL of filtered distilled water for 10 s in order to promote hemolysis, followed by the addition of 40 μL of 10 × filtered PBS. The centrifugation procedure, followed by the hemolysis procedure, was repeated three times. Splenic samples from each group were plated in pools using 6-well microplates containing 5 ml of RPMI 1640 culture medium (LGC Biotecnologia), supplemented with 0.05 mM β-mercaptoethanol (Sigma-Aldrich), 10% FBS (Cultilab) and 1% penicillin/streptomycin antibiotics (Vitrocell Embriolife). After 2 h of cultivation, the supernatant from each well was collected and transferred to a new plate. The viability of the splenocytes were evaluated using 0.4% trypan blue (Life Technologies) and the splenic samples were kept in an incubator overnight until the co-culture assay.

The cell lines utilized in this study included the human triple-negative breast adenocarcinoma MDA-MB-231 (ATCC^® HTB-26™) and human triple-negative breast carcinoma MDA-MB-453 (ATCC^® HTB-131™), both commercially obtained from the American Type Culture Collection. Additionally, the normal human fibroblast cell line FN1 (CAPPesq HCFMUSP No. 921/06) was isolated by Professor Dr. Durvanei Augusto Maria and its corresponding record deposited with CAPPesq at the Hospital das Clínicas of the University of São Paulo, under the mentioned registration number. The cells were cultured in RPMI-1640 medium (LGC Biotecnologia, Cotia, SP, Brazil), supplemented with 10% fetal bovine serum, 100 units/mL of penicillin G, and 100 μg/mL of streptomycin. Cell incubation took place at 37 °C in a CO₂-injected incubator.

PBMCs from CL and CL + DM were plated (2 × 10⁶) on 24-well tissue culture plates (TPP, Trasadingen, Switzerlan) using 13 mm round glass coverslips (Perfecta). Plates were incubated at 37°C under 5% CO₂ for 30 min. Non-adherent cells were removed by washing and adherent cells were cultured in RPMI 1640 medium (LGC Biotecnologia, São Paulo, Brazil) with 10% autologous serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 50 ng/mL M-CSF for 7 days. Glucose was added to cell cultures at the identical concentrations measured in the blood of each patient.

Mice peritoneal macrophages obtained after stimulation with thioglycolate for 3 days were harvested in RPMI 1640 medium (LGC Biotecnologia, São Paulo, Brazil), 20 μg/mL of gentamycin and were plated onto 13 mm² coverslips inside 24-well plates for adherence for 2 h at 37°C, 5% CO₂. Non-adherent cells were removed, and the macrophages were incubated overnight in RPMI supplemented with 10% FCS, as described above. Adhered macrophages were infected with L. amazonensis promastigotes (stationary growth phase) at a 10:1 parasite/macrophage ratio and incubated for 1 h at 34°C, 5% CO₂. Free parasites were washed out with 0.01 M phosphate buffered saline (PBS), and the cultures maintained for 24 h at 37°C, 5% CO₂. Infected macrophage cultures were treated with different concentrations of the analogs for an additional 24 h at 37°C, 5% CO₂. Monolayers were then washed with PBS at 37°C, fixed in methanol, and stained with Giemsa. The number of amastigotes and the percentage of infected macrophages were determined by counting at least 200 cells in duplicate cultures. Endocytic indices were obtained by multiplying the percentage of infected macrophages by the mean number of amastigotes per infected macrophage. The results were expressed as the percentage of surviving macrophages based on endocytic indices of treated and untreated macrophages as reported previously [19 (link)].

BMDMs were collected, plated and incubated as described in a previously study (58 (link)). Thereafter, macrophages were incubated with 100 nM of let-7e-5p inhibitor or the negative control (Ambion, Carlsbad, CA, USA) after a previous incubation with 3 μL of the FugeneHD transfection reagent (Roche, Madison, WI, USA) in 500 μL of RPMI 1640 medium (LGC Biotecnologia, São Paulo, SP, Brazil), supplemented as described above, for 20 min at room temperature. After 24 h of transfection, the cells were infected with L. amazonensis promastigotes, as described above.

Bone marrow from femurs and tibiae of C57BL/6 mice were obtained according to the protocol established by Inaba et al., (19 (link)). For differentiation in DCs, the cells were cultured in RPMI 1640 medium (LGC, biotechnology, Brazil) supplemented with 10% (v/v) fetal bovine serum (LGC, biotechnology, Brazil) 1% (v/v) penicillin/streptomycin (Gibco, USA), 30 ng/ml of GM-CSF and 15 ng/ml of IL-4 (Invitrogen, USA). The culture medium was changed on the third and fifth days. On day 8, differentiated dendritic cells were obtained. For differentiation in Mφ, the cells were cultured in R20/30 medium (RPMI 1640, 20% (v/v) fetal bovine serum and 30% (v/v) supernatant of L929 cells) (20 (link)). The medium was changed on the fourth day and, on the seventh day, macrophages were obtained. All cells were incubated in a humidified CO₂ incubator at 37°C. The cell phenotype was confirmed by BD FACS Fortessa flow cytometer as described below.