*3.6. Antioxidant Activity*

The antioxidant activity was evaluated by Trolox Equivalent Antioxidant Capacity (TEAC) method adapted for 96-well microplates and Infinite M200 (Tecan, Männedorf, Switzerland), using the radical cation ABTS•<sup>+</sup> and Trolox (Hoffman-La Roche) as standard [104,105]. Briefly: 10 μL of each sample was added to 200 μL of ABTS•<sup>+</sup> solution, were stirred and the absorbance at 734 nm was read at 6 min [11,106]. Trolox was used as standard and was assayed under the same conditions of the samples. Results were expressed as nmol of Trolox Equivalents per mg of contained proteins (nmol TE/mg protein).

### *3.7. Proteins SDS-PAGE Analysis*

Total jellyfish proteins and polypeptides fractions obtained from soluble proteins extracted with PBS were analyzed by SDS-PAGE. A FastCast premixed acrylamide solution 12% was used to prepared gels and "All Blue Precision Plus Protein Standard" (Biorad) was used as molecular weight marker. In order to visualize protein bands, gels were both analyzed by stain-free system with high sensitivity imagined using ChemiDoc ™ MP Imaging System (Biorad) and stained with Coomassie Brilliant Blue G-250 (Bio-Rad Protein Assay).

### *3.8. HEKa Cell Culture*

Human epidermal keratinocytes, isolated from adult skin (HEKa) were obtained from Cascade BiologicsTM (Gibco®) and routinely grown in EpiLife® medium with 60 μM calcium (GIBCO) as described in Leone et al. [10]. Trypan blue dye exclusion and automated counting method by Countess ™ was used for routinely cell viability assay and live cell counting. For all experiments, 0.15 × 10<sup>6</sup> cells/well (75000 cells/mL) were incubated in flat bottom 96-well microplates.

### *3.9. Cell Treatments and Oxidative Stress Induction with H2O2*

All jellyfish protein fractions were diluted in EpiLife® culture medium to reach a final concentration on the cells ranging from 0.05 and 20 μg/mL of proteins/peptides. Soon after dilution, the jellyfish samples were added to cells grown for 24 h in 96-well microplates at 37 ◦C with 5% CO2 (Thermo Forma direct heat CO2 incubator). Controls were included in each experiment and in each microplate with medium only (without cells), cells with only medium, and cells with the vehicle

(PBS or digestion buffers), at the same final concentration as in the cells treated with the jellyfish samples. For each independent experiment, each treatment, namely each sample, each control and each concentration, was replicated in five technical replicates. Microplates were then incubated for 24 h at 37 ◦C with 5% CO2 (Thermo Forma direct heat CO2 incubator).

### 3.9.1. Cell Treatments with Heat-Denatured Protein

Aliquots of soluble extracted proteins (SP) and the sub-fraction SP > 30 were also heat-denatured by heating at 100 ◦C for 10 min (Heat Treatment 100 ◦C) in a water bath, cooled, diluted in EpiLife® culture medium and administrated to the cells.

### 3.9.2. Cell Treatments with H2O2

In the experiments for antioxidant activity assay, HEKa cells (0.15 × 10<sup>6</sup> cells/well) were grown for 24 h to reach 80% of confluence in flat bottom 96-well microplates, and then were treated with the collagen peptides fractions from jellyfish and from bovine collagen at the same concentrations. Two controls with medium and vehicle were also included. After 24 h, 100 μL medium contained H2O2, at the final concentration of 0.1 mM were supplied and cells were incubated for 1 h at 37 ◦C with 5% CO2, as reported in Figure 7. Cell viability was assayed by MTS assay soon after the 1 h of treatment.

### *3.10. Cell Viability Assay*

MTS Cell viability test was used to establish the effects of the extracted jellyfish compounds. MTS assay was performed using CellTiter 96® AQueous One Solution Reagent (Promega) according to the manufacturer's instructions. 20 μL of CellTiter 96® AQueous One Solution Reagent were added to each well, the microplates were incubated for 90 min at 37 ◦C with 5% CO2 (Thermo Forma direct heat CO2 incubator) and the absorbance was read at 490 nm with Infinite M200. Data were expressed as percentage of the respective controls.
