*4.4. HUVECs Culture and Treatment*

HUVECs were cultured in 37 ◦C and 5% CO2 incubation, using EGM™-2 Medium Kit. The cells were subcultured and harvested using a 0.025% trypsin-EDTA solution. For experimental design, only HUVECs at passages 3–5 were used. They were then seeded in a 96- or 24-well plate, or 60 mm2 dishes. Sample peptides were added for the pretreated group following 2 h incubation before being challenged with 600 μM H2O2 for 24 h. The control group was cells without peptides treatment and H2O2 exposure.

## *4.5. Cell Viability Assessment*

Cytoprotective effect was evaluated by monitoring cell viability using MTT assay to HUVECs in a 96-well plate with 1 × <sup>10</sup><sup>4</sup> cells/well density. For further confirmation of the cytoprotective effect, live and dead cell assay was also performed. HUVECs were rinsed with warmed PBS in a 24-well plate with 2 × <sup>10</sup><sup>4</sup> cells/well, and then double stained using calcein-AM and PI 2.5 μM and 5 μM, respectively, following 30 min incubation at 37 ◦C. Stained cells were distinguished as live and dead cells under a fluorescence microscope (Leica DMI6000 B, Wetzlar, Germany).

#### *4.6. Determination of Intracellular ROS*

ROS generation in cells was detected in a 96-well black plate for quantification and a 24-well plate for microscopic observation. Pretreated cells were mixed with Hank's Balanced Salt Solution contained 20 μM DCFH-DA followed by 20 min incubation (37 ◦C). Fluorescence intensity was measured to determined intracellular ROS level at 485 and 528 nm (excitation and emission) (GENios, TECAN, Männedorf, Switzerland) and visually observed under fluorescence microscope (Leica DMI6000 B, Wetzlar, Germany).

#### *4.7. Nrf2 Nuclear Translocation Assessment*

To observe Nrf2 nuclear translocation, HUVECs culture was mixed with 3.7% paraformaldehyde in PBS for 15 min, and then permeabilized using 0.1% Triton X-100 dissolve with PBS (10 min), before being blocked in 2% bovine serum albumin with 30 min incubation. Later, anti-Nrf2 antibody with 1:200 dilution was added. After overnight incubation at 4 ◦C, the secondary antibody (Alexa Fluor® 488, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added (1:500 dilution with 1h incubation). To counterstain the nuclei, 2 μg/mL Hoechst 33342 was added following 10 min incubation. Visual observation was performed under fluorescence microscope (Leica DMI6000 B, Wetzlar, Germany).
