*3.1. Chemicals and Culture Media*

The Dulbecco's modified Eagle's medium (DMEM) (LM-D1109), fetal bovine serum (FBS) (FB-1001), trypsin-EDTA 0.05% (*w*/*v*) in PBS without (*w*/*o*) calcium and magnesium with phenol red (LM-T1705), Dulbecco's phosphate-buffered saline (PBS) *w*/*o* calcium and magnesium (LM-S2041), HEPES buffer (LM-S2030/100) and penicillin/streptomycin (P/S) (LM-A4118) were purchased from Biosera (Nuaillé, France). RPMI 1640 GlutaMAX (LMR 1640/500), DMEM GlutaMAXTM (21885025), DMEM/F12 with GlutaMAXTM supplement (31331028), MEM Alpha Media (22561-021) and Horse Serum (HS) heat-inactivated New Zealand origin (26050088) were obtained from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). The Mammary Epithelial Cell Growth Medium BulletKit was from Promega (C21110, Madison, WI, USA). Sodium pyruvate solution 100 mM (SH30239.01), L-Glutamine 200 mM (SH30034.01) and non-essential amino acids (SH30238.01) were purchased from Hyclone (Logan, UT, USA). Thiazolyl blue tetrazolium bromide (MTT, M5655) and dimethyl sulfoxide for cell culture (DMSO, D2650) were from Sigma-Aldrich (Darmstadt, Germany). The Vialight Plus Assay Kit used for the determination of cell viability was purchased from Lonza (Basel, Switzerland). The Cell Proliferation Kit III (EdU-488; FM) was from Promocell (PK-CA724-488FM, Heidelberg, Germany). Annexin V-FITC (#640945) and propidium iodide (PI) (#421301) were from Biolegend (San Diego, CA, USA). Hoechst was from Thermofisher (H3570, Waltham, MA, USA). Bovine Pituitary Extract (BPE), epidermal growth factor (EGF), insulin and hydrocortisone were purchased from Promega (C21110, Madison, WI, USA).

#### *3.2. Isolation and Characterization of OOPs*

The OOPs used in this study were purified by the methodology described in the sections below. The identity and purity of all the isolated compounds were confirmed using NMR spectroscopy with a Bruker Avance DRX 400 MHz. The 1H NMR spectra of the isolated OOPs are presented in Figure S1 and the NMR data in Table S4. The 1H NMR spectra were processed using either the MNova 6.0.2 (Mestrelab Research) or the TOPSPIN 4.1.4 software (Bruker, Billerica, MA, USA). The MS spectra and the optical rotation values are presented in Figure S7 and Table S5 respectively. The purified compounds were dissolved in DMSO and stored at −20 ◦C until use.

#### *3.3. Isolation of Oleomissional (6a,b,c) from Unripe Intact Olive Fruits*

Oleomissional (**6a,b,c**) was isolated from unripe (green) intact olive fruits collected in September–October. More specifically, 15 Kg of intact olive fruits (Lianolia Corfu variety) were immersed into 15 L of CH2Cl2 for thirty minutes and then the liquid phase (15 L) was collected and evaporated to dryness under vacuum in a rotary evaporator affording 80 g of a mixture which comprised mainly oleomissional (**6a,b,c**) and triterpenes. After that, 2.8 L of distilled water (pH = 6) was added, and the mixture was agitated for one hour. The mixture was heterogeneous and comprised the aqueous liquid phase and the solid terpenes, mainly oleanolic and maslinic acid, which could not be dissolved in water. The mixture was filtered, and the liquid aqueous phase (2.8 L) was collected and further extracted with 5.6 L of EtOAc. The organic phase was collected and evaporated to dryness under vacuum. The residue consisted of oleomissional (**6a,b,c**) (15 g) with >95% purity as measured by 1H-NMR in CDCl3 and NMR data in accordance with those previously described [34].

#### *3.4. Conversion of Oleomissional (6a,b,c) to Closed-Type Oleuropein Aglycone (3a,b)*

Oleomissional (**6a,b,c**) (3.5 g) isolated using the procedure described above was added to 2.5 L of water which contained NaHCO3 for the adjustment of pH to 7.8. After 24 h stirring at room temperature, the mixture became a homogenous solution that was further subjected to extraction by EtOAc (5 L). The organic phase was collected, washed with distilled water and evaporated to dryness under vacuum. The residue consisted of pure closed-type oleuropein aglycone (mixture of two isomers (**3a** and **3b**) as revealed by 1H-NMR in CDCl3 and NMR data in accordance with those previously described [35].

#### *3.5. Isolation of Oleacein (2) from Olive Tree Leaves*

Freshly dried olive tree leaves (1 Kg of Kalamon variety) with moisture content <10% (*v*/*v*) were mixed with water (10 L) at 25 ◦C and cut into small pieces in the presence of water using a blender. The mixture was allowed to stand for 30 min. Then, it was filtered, and the aqueous phase was collected and extracted with EtOAc (8 L). The organic phase was collected and evaporated using a rotary evaporator under reduced pressure affording a viscous liquid (10 g) containing oleacein (**2**) (purity 95% (*w*/*w*)) with NMR data in accordance with those previously described [98].

#### *3.6. Isolation of Oleocanthal (1) from Olive Oil*

Olive oil (10 kg) produced from olives of Kalamon variety, specifically selected to contain only oleocanthal (**1**) (1 g/kg) without other phenols, was mixed with distilled and deionized water (10 L) and stirred mechanically for 24 h. Subsequently, the mixture was left to stand for 24 h and the two layers were separated by gravity. The heavier aqueous layer was collected, filtered to remove insoluble substances, and re-extracted with EtOAc (1 L). The organic layer was collected and evaporated, affording oleocanthal (**1**) 7.5 g (purity > 95%) with NMR data in accordance with those previously described [98].

#### *3.7. Isolation of Ligstroside Aglycone (4a,b) and Oleocanthalic Acid (7)*

Both compounds were isolated from olive oil extracts as previously described by Karkoula et al. [35] and Tsolakou et al. [40].

#### *3.8. Cell Lines, Cell Culture Conditions and Treatment Protocols with OOPs*

The human breast cancer cell line MDA-MB-231 was kindly donated by Dr. P. Lymberi (Hellenic Institute Pasteur, Athens, Greece) and the MCF-7 cell line by Dr. Kletsas (Institute of Biosciences and Applications, NCSR Democritus, Athens, Greece). The lung cancer cell lines H1437 and H1299 were kindly provided by Prof. E. Kolettas (University of Ioannina Medical School & IMBB, Ioannina, Greece). The human gastric cancer cell lines MKN-45 and AGS as well as the human colon cancer cell lines HT-29 and Caco-2 were donated by Dr. D. Sgouras (Hellenic Institute Pasteur, Athens, Greece). The Huh-7 cells (Registry No. JCRB0403) were kindly provided by Prof. R. Bartenschlager (Heidelberg University, Germany) [99]. The HepG-2 hepatocarcinoma cells were a gift from Professor George Notas (University of Crete School of Medicine and University Hospital of Heraklion Emergency Department) and the PANC-1 human pancreatic epithelioid carcinoma cells from Dr. Ioannis Papasotiriou (Research Genetic Cancer Centre, RGCC SA, Florina, Greece). The non-tumorigenic epithelial cell line MCF 10A, and the human skin melanoma cell lines SK-MEL-28 and A2058 were purchased from ATCC (Manassas, VA,USA), while the human immortalized keratinocytes HaCaT were from CLS (Eppelheim, Germany) (https://www.clsgmbh.de/p800\_HaCaT.html, accessed on 6 August 2019). The NHDF human dermal fibroblasts isolated from human adult skin were obtained from Dr. Sophia Letsiou (APIVITA R&D department, Athens, Greece), the human mesenchymal stem cells derived from umbilical cord (Wharton's jelly stem cells (WJSCs)) were provided by Dr. Zoumpourlis (Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), Athens, Greece) and the MRC-5 human fetal lung fibroblasts from Dr. Vassilis Aidinis (Institute of Immunology, Biomedical Sciences Research Center Alexander Fleming, Athens, Greece).

All cell lines were incubated at 37 ◦C and 5% (*v*/*v*) CO2 and supplemented with 1% (*w*/*v*) antibiotic (final concentrations 100 U/mL penicillin and 100 μg/mL streptomycin). MDA-MB 231, MCF-7, SK-BR-3, A2058, NHDF, Huh-7 and AGS were cultured in high glucose (4500 mg/L) DMEM supplemented with 10% (*v*/*v*) FBS, 1 mM sodium pyruvate and non-essential amino acids, according to the manufacturer's recommendations (Dilution 1:100)—henceforth called "DMEM complete medium". The HaCaT cells were cultured in the same above-mentioned medium without the addition of sodium pyruvate and nonessential amino acids. Caco-2 cells were maintained in DMEM high glucose with 20% (*v*/*v*) FBS supplemented with 1 mM sodium pyruvate. PANC-1 cells were cultured in DMEM high glucose with 10% (*v*/*v*) FBS and 2 mM glutamine. SK-MEL-28, ME-180, H1437 and H1299 cells were cultured in RPMI GlutaMAX with 10% (*v*/*v*) FBS and 1 mM sodium pyruvate, while MKN-45 and HT-29 were maintained in RPMI GlutaMAX just with 10% (*v*/*v*) FBS. DMEM GlutaMAX with 10% (*v*/*v*) FBS plus 1 mM sodium pyruvate was used for the Hela and HepG-2 cell lines. MCF 10A was cultured in Mammary Epithelial Cell Growth Medium supplemented with 5% (*v*/*v*) HS, 0.004 mL/mL BPE, 10 ng/mL EGF, 5 μg/μL insulin and 500 ng/mL hydrocortisone. WJSCs were cultured in DMEM/F12 GlutaMAXTM supplemented with 10% (*v*/*v*) FBS, non-essential amino acids (Dilution 1:100) and HEPES buffer at a final concentration of 15 mM. Finally, the MRC-5 cell line was cultured with MEM Alpha Medium with 10% FBS.

The OOPs tested in this study were first dissolved in an appropriate DMSO volume such as to prepare a final stock solution of 50 mM, which was further used to prepare various compound concentrations in DMSO-based solutions. The final DMSO concentration in the cell culture was maintained constant in all treated groups of any given experiment and never exceeded the value of 0.2% (*v*/*v*). For the treatment experiments, cells were plated in 96-well culture plates, in 100 μL of complete medium at a seeding density of 5000–10,000 cells/well—depending on cells' size and doubling time—and were allowed to adhere overnight (~16 h). The next day, 150 μL of complete medium was added to each well and the cells were treated with various concentrations of OOPs. Each concentration was tested in triplicate and was repeated 2–3 times. To prepare the final concentration solutions, 0.5 μL of a 500× stock of each tested compound in DMSO was added directly into each well in a final volume of 250 μL culture medium and was gently mixed by pipetting. Control wells were prepared under the same experimental conditions by adding only DMSO at a final concentration of 0.2% (*v*/*v*). Compounds were not renewed during the entire period of cell exposure. All cells were cultured at 37 ◦C in a 5% (*v*/*v*) CO2 for 24, 48 or 72 h.
