Mem earle medium

Three cell lines were used, HUVECs, HaCaT and HeLa cells. The HUVEC cell line was cultured in MEM/Earle medium (Cultilab, Brazil), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil), 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA), and 1% l‐glutamine (200 μm) (Sigma‐Aldrich, St Louis, MO, USA). The HaCaT cell line was cultured in MEM/Earle medium (Cultilab), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab), 1% antibiotic/antimycotic (Invitrogen), 1% l‐glutamine (200 μm) (Sigma‐Aldrich), 1% non‐essential amino acids (10 mm; Sigma‐Aldrich), and 1% sodium pyruvate (100 mm; Sigma‐Aldrich). The HeLa cell line were cultured in MEM/Earle medium (Cultilab), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab), 1% antibiotic/antimycotic (Invitrogen) 1% l‐glutamine (200 μm; Sigma‐Aldrich), and 1% non‐essential amino acids (10 mm; Sigma‐Aldrich). A total of 10⁶ cells from each cell line were seeded in 75 cm² culture flasks and kept at 37 °C in an atmosphere of 5% CO₂.
We did all the experiments with another cervix cell line (SiHa – ATCC, Manassas, VA, USA) treated with the peptide, but without the co‐treatment with conditioned medium of endothelial cells (HUVECs), and the results were the same with the HeLa; for this reason we did not continued with the co‐treatment.

The annexin A1-derived peptide Ac_2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK)^{83 (link)} was obtained from THERMO SCIENTIFIC (Waltham, MA, USA) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration in culture medium of 1uM. Piplartine (C₁₇H₁₉NO₅; CAS number 20069-09-4) was isolated as previously described^{36 (link)} and dissolved in DMSO at a final concentration in culture medium of 10 or 20 µM. PL was also dissolved in absolute ethyl alcohol for UV–Vis absorbance and fluorescence spectroscopy assays. Ac_2-26 and PL concentrations were determined by experiments previously performed by our group^{20 (link),84 (link)} and by other authors^{85 (link)–87 (link)}. Bacterial lipopolysaccharide (LPS, Escherichia coli O127:B8, SIGMA ALDRICH, Poole, Dorset, UK), a component of the surface membrane of most gram-negative bacteria and potent stimulator of immune cells, was diluted in 10% MEM-Earle medium (CULTILAB, Campinas, SP, Brazil) at a final concentration in culture medium of 10 µg/mL.

The Hep-2 cell line, which was originally established from an epidermoid carcinoma of the larynx, was used in the present study (ATCC, Rockville, Maryland, USA). Cell line authentication was performed using the AmpFLSTR Identifiler PCR Amplification Kit (Life Technologies) at the Special Techniques Laboratory, Hospital Israelita Albert Einstein (LATE -HIAE), Sao Paulo. We found 100% identity of our cell line compared with the American Type Culture Collection (ATCC) database. The cells were seeded at a density of 2×10⁶ cells in a 75-cm² culture flask (Corning, NY, USA) and incubated in MEM-Earle medium (Cultilab, Campinas, SP, Brazil), pH 7.5, supplemented with 20% fetal calf serum (Cultilab), 1% non-essential amino acids, and 0.1% antibiotic/antimycotic solution (Invitrogen Corporation, Carlsbad, CA, USA), at 37°C in a humid atmosphere of 5% CO₂[43] (link).