*2.3. OMW Acquisition and Preparation*

The extract was obtained following the method reported by De Marco et al., with some modifications [18]. Two liters of olive oil mill wastewater (OMW) were acidified to pH 2 with HCl and washed three times with hexane (1:1, *v*:*v*) in order to remove the lipid fraction. The mixture was vigorously shaken and centrifuged under 3000 rpm for 3 min at 10 ◦C. The phenolic compounds were extracted by mean of ethyl acetate three times in a separating funnel (1:4 *v*:*v*), and then the combined extracts were centrifuged for 5 min at 3000 rpm at 10 ◦C. The organic phase was separated and filtered through a sintered glass Buchner apparatus. Then the ethyl acetate was evaporated under vacuum using a rotary vacuum evaporator at 25 ◦C (headspace analysis was performed). Finally, the dry residue was again dissolved in 100 mL of water, filtered using PTFE 0.45 μm (diameter 15 mm) syringe filter, and stored at 4 ◦C until subsequent analyses. An aliquot of the obtained extract (called L) was freeze-dried in a VirTis lyophilizer (Gardiner, NY, USA), and the obtained sample was called S. For all the experiments, we used both types of phenolic extracts, the freeze-dried (S) and the wet (L) type.

#### *2.4. Determination of Total and Individual Phenolic Content of OMW Polyphenolic Extracts*

The total phenol content was determined spectrophotometrically as previously described by Bruno et al. with some modifications [19]. An aliquot portion (0.1 mL) of phenolic extract was placed in a 25 mL volumetric flask and mixed with 20 mL of deionized water and 0.625 mL of the Folin–Ciocalteau reagent. After 3 min, 2.5 mL of saturated solution of Na2CO3 (20%) was added. After that, the mixture was incubated for 12 h at room temperature and in the dark. The sample's absorbance was measured at 725 nm against a blank and compared with a gallic acid (GA) calibration curve (concentration between 1 and 10 mg L<sup>−</sup>1). The results were expressed as mg of GA g−<sup>1</sup> of phenolic extract.

Identification and quantification of S and L extracts' main phenolic compounds were performed by HPLC-DAD (Dionex Ultimate 3000 RSLC, Waltham, MA, USA), as previously described [20]. The phenolic determination was conducted using the Dionex Acclaim 120 C18 analytical column (3 μm, 150 × 3 mm) (Thermo Scientific, Waltham, MA, USA) set at 35 ◦C, a flow rate of 1 mL min<sup>−</sup>1, and an injection volume of 5 μL. Water/acetic acid (98:2, *v*/*v*) (A) and acetonitrile (B) were used as mobile phases, and the applied gradient was the following: 95% A and 5% B (5 min), 80% A and 20% B (10 min), 75% A and 25% B (15 min), 65% A and 35% B (20 min), 0% A and 100% B (25 min), and 95% A and 5% B (35 min). Quantification was performed by pure standard (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and data were expressed as mg g−<sup>1</sup> of phenolic extract.

#### *2.5. Cell Culture*

Human umbilical vein endothelial cells (HUVECs) were acquired from Cell Application (San Diego, CA, USA) and grown as previously described [21]. Briefly, cells were grown in endothelial cell basal medium supplemented with cell growth supplement (EGM-V2 # 213K-500) as per company instructions. When confluent, cells were sub-culture at a split ratio of 1:2 and used within three passages. Unless specified in the text, cells were plated in 96-well plates (Corning, Lowell, MA, USA) at a concentration of 10<sup>5</sup> cells/mL and processed for experiments in a complete medium containing the different concentrations of the extracts.

Human pulmonary artery smooth muscle cells (HPASMCs) were acquired from Cell Application (San Diego, CA, USA) and grown as previously described [22]. Briefly, cells were grown in smooth muscle cells basal medium supplemented with cell growth supplement (HSMCs-kit #311K-500) as per company instructions. When confluent, HSMCs were sub-culture at a split ratio of 1:2 and used within three passages. Unless specified in the text, cells were plated in 96-well plates (Corning, Lowell, MA, USA) at a concentration of 10<sup>5</sup> cells/mL and processed for experiments in a complete medium containing the different concentrations of the extracts.

According to data obtained in our previous studies [23–26], we decided to test the extracts at the doses of 10, 25, 50, and 100 μg/mL.

#### *2.6. Cell Metabolic Assay*

Cell metabolic activity was evaluated using the oxidizable and reducible colorimetric probes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [23]. Cells were treated as indicated in figure legends and then processed for the MTT assay. After treatments, cells were added with 20 μL MTT solution (5 mg/mL) in medium M199 and placed at 37 ◦C in a cell incubator for 4 h. After that, the medium was discarded, and the converted dye was solubilized with acidic isopropanol (0.04 N HCl in absolute isopropanol), and the multi wells were read at 570 nm using a GENios plus microplate reader (Tecan) with background subtraction at 650 nm. Results were expressed as a percent of untreated control cells. Cell viability was calculated by the following equation: Cell viability (%) = (Abs of sample/Abs of control) × 100, where Abs of sample is the absorbance of the cells incubated with the different concentrations of the two extracts, and Abs of control is the absorbance of the cells incubated with the culture medium only (positive control).

#### *2.7. Measurement of Intracellular ROS*

Intracellular ROS levels were determined by using the ROS molecular probe 2 ,7 dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probe, Eugene, OR, USA) as previously described with minor modification [24,25]. In this assay, ROS oxidize H2DCF, producing the fluorescent compound DCF, the fluorescence levels of which are proportional to the amount of intracellular ROS. Cells were treated as indicated in figure legends and then processed for the intracellular ROS assessment. For the ROS assay, cells were incubated for 30 min with Hank's Balanced Salt Solution (HBSS) containing 5 μM H2DCF-DA, then washed twice with HBSS, and then fluorescence was measured by using a GENios plus microplate reader (Tecan, Mannedorf, Switzerland. Excitation and emission wavelengths used for fluorescence quantification were 485 and 535 nm, respectively. All fluorescence measurements were corrected for background fluorescence and protein concentration. Using untreated cells as a reference, the antioxidant and prooxidant outcomes were evaluated by comparing five measurements and expressed as a percentage of untreated control cells.

#### *2.8. Cell Viability Assay*

Cell viability was assessed as previously described [26] by using the CytoTox-ONE™ (Promega, Madison, WI, USA) kit. The CytoTox-ONE™ homogeneous membrane integrity assay is a fluorometric method for estimating the number of nonviable cells present in multi-well plates. The assay measures the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. Then the LDH released into the culture medium is measured with a 10-min coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of dead cells with the lysed membrane.

Cells were treated as indicated in figure legends and then processed as per company instructions at each experimental point's end. Fluorescence was measured by using a GENios plus microplate reader (Tecan, Mannedorf, Switzerland). Excitation and emission wavelengths used for fluorescence quantification were 535 and 620 nm, respectively. All fluorescence measurements were corrected for background fluorescence and protein concentration, and results were expressed as a percentage of untreated control cells.

#### *2.9. Statistical Analysis*

Data were expressed as means ± S.D. of the indicated number of experiments. Oneway analysis of variance (ANOVA) followed by a post-hoc Newman–Keuls multiple comparison test were used to detect differences of means among treatments with significance defined as *p* < 0.05. Statistical analysis was performed using GraphPad Prism version 8.00 for Windows, GraphPad Software, San Diego, CA, USA.
