*3.13. Cell Line Cultures*

In this study, four cell lines were used: three of them tumoral, such as the human breast adenocarcinoma cell line (MCF-7, ATCC, Manassas, VA, USA), human leukemia cell line (HL-60 ATCC), human lung cancer cell line (NCI-H460, ATCC) and an immortalized human gingival fibroblast-1 cell line HGF-1 (ATCC CRL-2014). MCF-7 and HGF-1 cell lines were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) (Biowest, Barcelona, Spain) supplemented with 10% fetal bovine serum (Biowest, Barcelona, Spain), 1% penicillin-streptomycin solution 100X and 0.5% of amphotericin B (Biowest, Barcelona, Spain), while NCI-H460 cells were cultured in Roswell Park Memorial Institute (RPMI-1640) medium (Biowest, Barcelona, Spain) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution 100X and 0.5% of amphotericin B, and HL-60 cells were cultured in RPMI-1640 medium (Biowest, Barcelona, Spain) supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin solution 100X and 0.5% of amphotericin B (Biowest, Barcelona, Spain). Cells were maintained sub-confluent at 37 ◦C in humidified air containing 5% CO2. Cultured cells were collected by gentle scraping when confluence was reached 75% in the case of the MCF-7, HFG-1 and NCI-H460, as they are adherent cells. The scrapping of HL-60 cells was not performed, because these are cells in suspension. So, they were collected by centrifugation at 1500 rpm for 5 min.

### *3.14. Cytotoxic E*ff*ects on Tumor Cells Assay*

For cytotoxic e ffects on the tumor cells assay, HL-60, MCF-7 and NCI-H460 cell lines were incubated at 2 × 104, 8 × 10<sup>3</sup> and 8 × 10<sup>3</sup> cell lines per well, respectively in the presence of di fferent concentrations (19 – 1 × 10<sup>4</sup> μg mL−1) of EPS. The experiment was conducted individually with each cell line in a 96-well microplate (Sarstedt, Nümbrecht, Germany) for 72 h. The incubation conditions were as follows: temperature 37 ◦C, 5% CO2 and a humid atmosphere. As a control, the same cell lines were used without treatment. The proliferation of these cell lines was estimated by the MTT (Sigma-Aldrich, St. Louis, MO, USA) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [31]. Briefly, a volume of 10 μL of the MTT solution (5 mg mL−<sup>1</sup> in phosphate-bu ffered saline) was added to each well. The microplates were incubated at 37 ◦C for 4 h. The yellow tetrazolium salt of MTT was reduced by mitochondrial dehydrogenases of metabolically active viable cells to form insoluble purple formazan crystals. Formazan was dissolved by the addition of sulfated–isopropanol (150 μL of 0.04-N HCl–2 propanol) and measured spectrophotometrically at 550 nm (Micro Plate Reader 2001, Whittaker Bioproducts, Promega, Wisconsin, USA). The relative cell viability was expressed as the mean percentage of viable cells compared with untreated cells. Four samples for each tested concentration were included in each experiment. Measurements were carried out in triplicate independent experiments.

### *3.15. Cytotoxicity Assay in Healthy Cell Line*

For the cytotoxicity assay, HGF-1 cells were incubated at 1.5 × 10<sup>4</sup> cells per wells in the presence of di fferent concentration of EPS (19−1 × 10<sup>4</sup> μg mL−1) in a 96-well microplate (Sarstedt, Nümbrecht, Germany) for 72 h. The incubation conditions were as follows: temperature 37 ◦C, 5% CO2 and a humid atmosphere. Cells proliferation was estimated by the MTT (Sigma-Aldrich, St. Louis, MO, USA) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [31], as explained above.
