*2.2. Sea-Buckthorn Seed Oil Fatty Acid Composition*

Targeted quantification of fatty acids was carried out by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Analyses were performed on a triple quadrupole mass spectrometer model API3200 (Sciex) coupled with an Infinity 1260 binary pump (Agilent) and autosampler. Analyst software version 1.5.2 was used for data acquisition and processing. After testing several stationary phases, chromatographic separation was achieved on a Phenomenex Luna pentafluorophenyl (PFP2) column (100 mm × 2 mm, 3 µm, 100 Å) using a mobile phase composed of water and acetonitrile at a flow rate of 0.3 mL/min. Seed oil was dissolved in ethanol at 1 mg/mL, and 3 µL of the solution were injected in the LC-MS/MS system. The mass spectrometer was operated in negative electrospray ionization (ESI). Acquisitions were carried out in multiple reaction monitoring (MRM) mode using the specific traces of each fatty acid (see Supplementary information Table S1). A general screening of the seed oil volatile constituents was performed by gas chromatography–mass spectrometry (GC/MS). A Perkin Elmer CLARUS 680 gas chromatograph (equipped with an ELITE 5 MS column (1,4-bis (dimethylsiloxy)) phenylene dimethyl polysiloxane stationary phase, 30 m, 0.25 mm ID, 0.25 µm film thickness, eluted with helium at 1 mL/min), connected to a Clarus SQ8T quadrupole, was used for this purpose. For analysis, 1 µg of seed oil solutions prepared at 50 mg/mL in ethanol were injected in split mode (40:1). Compound identification was achieved by searching the obtained mass spectra in the NIST and Wiley libraries.

## *2.3. Cell Proliferation and Toxicity Assays*

Cytotoxicity was assessed by cellular lactate dehydrogenase (LDH)release (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI, USA). Specifically, 10,000 cells were seeded in triplicate overnight in 96-well plates and incubated the next day with serial dilutions of stock solution for an additional 72 h. Two controls were included—a negative control (cell culture medium only) and a positive control (addition of Cell Lysis Reagent 10x, Promega, 45 min prior to LDH detection). For background subtraction, additional triplicates of cell-free wells were incubated with tested solutions. For LDH analysis, 50 µL of supernatant were collected from each well and incubated with assay reagent for 30 min in the dark. After the addition of stop solution, absorbance was read at 490 nm by using a microplate reader Anthos Zenyth 3100 (Wals, Austria). Cytotoxicity was assessed as a percentage of the positive control, according to the formula cytotoxicity = 100\*(Sample OD − background OD)/(average positive control − average background).

Proliferation was assessed by MTS assay (CellTiter 96 AQueous One Solution Reagent, Promega, Madison, WI USA). Specifically, 10,000 cells were seeded in triplicates overnight in 96-well plates and incubated the next day with cell culture medium and/or serial dilutions of stock solution for 72 h. A positive control (non-treated cells) was included. For background subtraction, additional triplicates of cell-free wells were incubated with tested solutions. On the day of the reading, the cell media was removed, and 100 µL of fresh medium and 20 µL of MTS reagent were added to each well. The plate was incubated at 37 ◦C for 3 h in a humidified, 5% CO<sup>2</sup> atmosphere. Absorbance was read at 490 nm by using a microplate reader Anthos Zenyth 3100 (Wals, Austria). Proliferation was assessed as a percentage of the control: % of proliferation = 100\*(sample OD − background OD)/(average control − average control background).

Oil Red staining: Cells were fixed in 3.7% formaldehyde for 30 min to 1 h, washed with water, and then incubated with 60% isopropanol for 5 min. Oil Red O Stock solution (Sigma-Aldrich, St Louis, MO, USA) was reconstituted with 100% isopropanol. Oil Red O Working solution was prepared 15 min before staining by mixing three parts of Oil Red O Stock solution and two parts of water, and then the solution was filtered through Whatman No.1 filter paper. The cells were stained with Oil Red O Working solution for 10 min at room temperature and afterward washed 2–5 times with water until no excess stain was seen. DAPI staining can be performed after this step for 10 min, at room temperature, and protected from light. The red lipid droplets were visualized by using fluorescent microscopy with a Nikon TI (Nikon, Tokyo, Japan) inverted fluorescence microscope.

Real-time impedance readings: Cell adhesion and cell proliferation were assessed using RTCA DP platform (Agilent Technologies, Santa Clara, CA, USA). Cells were preirradiated with UVA for 30 min as previously described [17], and then they were trypsinized and seeded in E-16 plates at a density of 20,000 cells/well, with or without the cell medium, and supplemented or not with sea-buckthorn oil 1/8000. Readings were collected every 5 min for 24 h. Cell index at 2 h (at plateau) was used for statistical analysis (One-Way Anova, Dunnett multiple comparison, where data were compared to control. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001)

Scratch-wound assay: Cells were seeded in a 35 mm glass-bottom dish and grown to confluence in normal cell culture conditions. A scratch was performed with a 200 µL pipette tip, with debris washed and cells further incubated in cell medium supplemented with 1/8000 sea-buckthorn oil for 24 h, in Nikon Biostation IM. Images were acquired with 20× Ph objective every 20 min, and data were analyzed with NIS-Elements BR (Nikon Tokyo, Japan).

#### *2.4. Evaluation of the Expression CD36*

Immunofluorescence (IF): Cells were grown on coverslips fixed with 4% formaldehyde for 5 min, rinsed in phosphate buffer saline (PBS), permeabilized in 0.1%Triton-X 100 and in 0.5% BSA for 10 min and incubated for 2 h with the primary antibody (anti-CD36 ThermoFisher PA1-16813, 1:100) at room temperature. After 3 × 5 min washes in PBS, cells were incubated with the secondary antibody (goat anti-rabbit Oregon Green 488, Invitrogen 011038, 1:2000 in PBS), and the nuclei were counterstained with DAPI. Image acquisition was performed with a Leica TCS SP8 white light laser confocal microscope.

Western blotting (WB): Cells were lysed in RIPA buffer with 1% protease inhibitor cocktail (P8340 Sigma), centrifuged for 10 min at 11,000 rpm (Ohaus R5510), and the supernatant boiled for 10 min (1:1 *v*/*v*) in Laemmli two times. Then, 40 µg of protein were loaded on each well, migrated in 10% SDS-PAGE gel for 2 h at 20 mA/gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (BioRad) (1 h, 100 V). The membrane was incubated overnight in primary antibodies (anti-CD36 AF2519, R&D Systems, anti-GAPDH PA1-987, ThermoFisher), washed three times in TBS-T 0.5% and one time in TBS the next day and incubated for 1 h at room temperature (RT) in secondary antibodies (chicken-anti goat sc2953, SantaCruz, goat-anti rabbit 92680011, LiCor). Image acquisition was performed using a c-Digit scanner (LI-COR).
