*2.1. Reagents*

MeDHICA was prepared as described in [42]. MeDHICA-melanin was prepared by aerial oxidation of MeDHICA in phosphate bu ffer at pH 8.5, as previously reported [42]. Phosphate bu ffer saline (PBS), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (HyClone), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-,7--dichlorodihydrofluorescein diacetate (H2DCFDA), l-glutamine, trypsin-EDTA, Triton, 5,5--dithiobis-2-nitrobenzoic acid (DTNB), thiobarbituric acid (TBA), and Bradford reagen<sup>t</sup> were from Sigma-Aldrich (St. Louis, MI, USA). Bicinchoninic acid (BCA) protein assay kit was from Thermo Scientific (Waltham, MA, USA). Antibodies against nuclear factor erythroid 2–related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1) were from Cell Signal Technology (Danvers, MA, USA). Antibodies against B-23 andβ-actin and the chemiluminescence detection system (SuperSignal ® West Pico) were from Thermo Fisher Scientific (Waltham, MA, USA).

## *2.2. Cell Culture*

Human immortalized keratinocytes (HaCaT) were from Innoprot (Derio, Spain). Cells were cultured in DMEM, supplemented with 10% fetal bovine serum, 2 mM l-glutamine and antibiotics in a 5% CO2 humidified atmosphere at 37 ◦C. Every 48 h, cells were refreshed in a ratio 1:5. The culture medium was removed and cells were rinsed with PBS and then detached with trypsin-EDTA. After centrifugation (5 min at 1000 rpm), cells were diluted in fresh medium.

#### *2.3. Analysis of Cell Viability*

Cells were seeded in 96-well plates (100μL/well) at a density of 2.5×10<sup>3</sup> cells/cm2. 24 h after seeding, cells were incubated with increasing concentrations (0.1, 1, 5 and 10 μg/mL) of MeDHICA-melanin. Mother solutions of MeDHICA-melanin were prepared in DMSO at a concentration of 0.2 mg/mL and proper aliquots were added to the incubation medium to ge<sup>t</sup> the desired concentration. After 24 h and 48 h incubation, cell viability was assessed by the MTT assay. The MTT reagent, dissolved in DMEM without phenol red, was added to the cells (0.5 mg/mL). After 4 h at 37 ◦C, the culture medium containing MTT was removed and the resulting formazan salt was dissolved in 2-propanol containing 0.01 M HCl (100 μL/well). Absorbance values of blue formazan were determined at 570 nm using an automatic plate reader (Microbeta Wallac 1420, Perkin Elmer, Milano, Italy). Cell survival was expressed as the percentage of viable cells in the presence of MeDHICA-melanin compared to the controls, represented by untreated cells and cells supplemented with identical volumes of DMSO, in order to exclude a possible e ffect of DMSO on cell viability.

#### *2.4. UVA irradiation and H2DCFDA Assay*

To evaluate the protective e ffect of MeDHICA-melanin against oxidative stress, cells were plated at a density of 3.5 × 10<sup>4</sup> cells/cm<sup>2</sup> (in 6o mm eukaryotic cell plates), pre-incubated in the presence of increasing concentration (0.1–10 μg/mL) of MeDHICA-melanin for di fferent lengths of time (from 5 to 120 min) and stressed by UVA light for 10 min (100 J/cm2) [45]. Then, cells were incubated with the cell permeable, redox-sensitive fluorophore H2DCFDA at a concentration of 20 μM for 30 min at 37 ◦C. Cells were then washed with cold PBS 2 times, detached by trypsin, centrifuged at 1000 rpm for 10 min and resuspended in PBS containing 30 mM glucose, 1 mM CaCl2, and 0.5 mM MgCl2 (PBS plus) at a cell density of 1 × 10<sup>5</sup> cells/mL. H2DCFDA is nonfluorescent until it is hydrolyzed by intracellular esterases, and in the presence of ROS it is readily oxidized to the highly fluorescent 2-,7--dichlorofluorescein (DCF). DCF fluorescence intensity was measured at an emission wavelength of 525 nm with excitation wavelength set at 488 nm using a Perkin-Elmer LS50 spectrofluorometer (Perkin Elmer, Milano, Italy). Emission spectra were acquired at a scanning speed of 300 nm/min, with 5 slit widths for excitation and emission. ROS production was expressed as percentage of DCF fluorescence intensity of the sample under test, with respect to the untreated sample.

#### *2.5. Western Blot Analysis*

HaCaT cells were plated at a density of 3.5 × 10<sup>4</sup> cells/cm<sup>2</sup> (100 mm eukaryotic cell plates) in complete medium for 24 h and then treated with 10 μg/mL of MeDHICA-melanin for 5, 15, 30 and 60 min. To extract nuclear proteins, cells were first incubated with PBS bu ffer containing 0.1% Triton and protease inhibitors to extract cytosolic proteins. After centrifugation at 1200 rpm for 10 min, nuclear pellet was obtained and proteins were extracted by resuspending the pellet in radioimmunoprecipitation assay bu ffer (RIPA) bu ffer (150 mM NaCl, 1% NP-40, 0.1% SDS, proteases inhibitors in 50 mM Tris-HCl pH 8.0). Proteins were quantified by BCA protein assay kit. Western blotting was used to analyze 100 μg of proteins, as reported [46]. Nuclear factor erythroid 2–related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1) levels were detected by using specific antibodies. To normalize protein intensity levels, specific antibodies against B-23 and β-actin were used for nuclear and cytosolic extracts, respectively. Signals were detected by using the chemiluminescence detection system.

## *2.6. Catalase Assay*

HaCaT cells were plated at a density of 3.5 × 10<sup>4</sup> cells/cm<sup>2</sup> (in a 100 mm eukaryotic cell plate) in complete medium for 24 h and then treated with 10 μg/mL of MeDHICA-melanin for 60 min. At the end of the experiment, total cell lysate was obtained by resuspending each cell pellet in 50 μL of lysis buffer (100 mM Tris-HCl, 300 mM NaCl and 0.5% NP-40 at pH 7.4 with addition of inhibitors of proteases and phosphatases). Proteins were quantified by BCA protein assay kit. To measure catalase activity, a procedure reported in the existing literature was followed [47]. Briefly, cell lysates (50 μg of proteins) were incubated for 30 min at room temperature in 1 mL of hydrogen peroxide solution (50 mM potassium phosphate buffer, pH 7.0, 0.036% *w*/*w* H2O2). Then, the hydrogen peroxide concentration in solution was determined by measuring the absorbance at 240 nm. The percentage of peroxide removed was calculated as following:

$$\% \,\text{H}\_2\text{O}\_2 \text{ reduced} = 1-\text{OD}\_{240\text{nm}} \,\text{sample}/\text{OD}\_{240\text{nm}} \,\text{standard}$$

Standard is referred to the hydrogen peroxide solution in the absence of lysate and measured at 240 nm after 30 min of incubation.

#### *2.7. Determination of Intracellular Glutathione (GSH) Levels*

HaCaT cells were plated at a density of 3.5 ×10<sup>4</sup> cells/cm<sup>2</sup> (60 mm eukaryotic cell plates) in complete medium for 24 h and then treated with 10 μg/mL of MeDHICA-melanin for 60 min. At the end of the photoirradiation experiment, cells were lysed, and protein concentration was determined by the Bradford colorimetric assay. Proteins (50 μg) were incubated in the presence of 3 mM EDTA, 144 μM DTNB in 30 mM Tris-HCl at pH 8.2, and centrifuged at 13,000 rpm for 5 min at 4 ◦C. Supernatants were collected, and the absorbance was measured at 412 nm by using a multiplate reader (Bio-Rad, Hercules, CA, USA). GSH levels were expressed as % of the sample under test with respect to the untreated sample.

#### *2.8. Analysis of Lipid Peroxidation Levels*

HaCaT cells were seeded at a density of 3.5 × 10<sup>4</sup> cells/cm<sup>2</sup> (100 mm eukaryotic cell plates) in complete medium for 24 h and then treated with 10 μg/mL of MeDHICA-melanin for 120 min. After UVA irradiation, cells were kept at 37 ◦C for 90 min, before performing the thiobarbituric acid reactive substances (TBARS) assay as described [48]. Briefly, cells were detached and 5 × 10<sup>4</sup> cells suspended in 0.67% TBA containing 20% trichloroacetic acid (TCA) (1:1 *v*/*v*). After heating for 30 min at 100 ◦C, samples were centrifuged at 2500 rpm for 5 min at 4 ◦C, and supernatants spectrophotometrically analyzed at 532 nm.

#### *2.9. Quantification of Internalized Melanin*

HaCaT cells were plated at a density of 3.5 × 10<sup>4</sup> cells/cm<sup>2</sup> (100 mm eukaryotic cell plates) in complete medium for 24 h and then treated with 10 μg/mL of MeDHICA-melanin for 60 min. After treatment, total cell lysate was obtained, 50 μg of proteins (Bradford assay) were diluted in 1 mL of potassium phosphate buffer (50 mM, pH 7.0) and UV-vis spectra were recorded. The amount of MeDHICA-melanin internalized by the cells was determined by using a calibration curve obtained with pureMeDHICA-melanin. In particular, increasing concentrations (0.6–20 μg/mL) of MeDHICA-melanin, alone or in the presence of 50 μg of cell lysate, were used to record the UV-vis spectra. The calibration curve was built by plotting values of absorbance at 330 nm against MeDHICA-melanin concentration.

#### *2.10. HPLC and LC-MS Analysis of Cell Lysate*

HPLC analysis was performed on an instrument (Agilent 1100, Santa Clara, CA, USA) equipped with a binary pump and a SPD-10AV VP UV-vis detector set at 300 nm. The chromatographic separation was achieved on a Sphereclone octadecylsilane-coated column, 250 mm × 4.6 mm, 5 μm particle size

(Phenomenex, Torrance, CA, USA) at 0.7 mL/min using binary gradient elution conditions as follows: 0.1% formic acid (solvent A), acetonitrile (solvent B) from 35% to 70%, 0–45 min. LC-MS analyses were run on a LC-MS ESI-TOF 1260/6230DA Agilent instrument operating in positive ionization mode in the following conditions: Nebulizer pressure 35 psig; drying gas (nitrogen) 5 L/min, 325 ◦C; capillary voltage 3500 V; fragmentor voltage 175 V. An Eclipse Plus C18 column, 150 × 4.6 mm, 5 μm (Agilent), at a flow rate of 0.4 mL/min was used, using the same eluant as above. The cell lysate, obtained as described in Section 2.9, was lyophilized and subjected to acetylation treatment with acetic anhydride (500 μL) and pyridine (75 μL) overnight. After repeated washings with methanol to remove solvents, the residue was taken up in methanol and analyzed by HPLC and LC-MS. A control lysate sample obtained in the absence of MeDHICA-melanin was also analyzed.
