*3.5. Immunocytochemistry*

Cells at a density of 1.0 × 105 cells/mL were seeded into a 4-well chamber slide. After incubation for about 16 h, cells were exposed to 20 ΐM fucoxanthin for a further 6 h. Subsequently, cells were fixed with 1% paraformaldehyde for 30 min and then washed three times with phosphate-buffered saline (PBS) for 5 min each time. Cells were permeabilized with PBS containing 1% Triton X-100 for 30 min and then washed with PBS. Cells were blocked with PBS containing 5% bovine serum albumin for 1 h at 37 °C, and then treated with an anti-Nrf2 antibody diluted in blocking medium (1:125 dilution) overnight. To visualize the primary anti-Nrf2 antibody, cells were treated with a FITC-conjugated secondary antibody (1:125) for 2 h. After washing with PBS, stained cells were mounted onto microscope slides in mounting medium containing DAPI (Vector, Burlingame, CA, USA). Images were collected using the LSM 510 program on a Zeiss confocal microscope. 

## *3.6. ChIP Assay*

Cells were processed using the SimpleChIP® enzymatic chromatin IP kit (Cell signaling technology, Beverly, MA, USA) according to the manufacturer's instructions. Briefly, proteins were cross-linked to DNA by adding 1% formaldehyde to the culture dishes and rocking on a platform for 10 min at room temperature. The cross-linking was stopped by the addition of glycine solution. Cells were washed twice with ice-cold PBS, pelleted by centrifugation, and resuspended in 1 mL cell lysis buffer containing protease inhibitors. Soluble chromatin was sheared by sonication and then centrifuged at 15,000× *g*. Diluted supernatants were pre-cleared and blocked with protein A/G agarose, and the sonicated chromatin-DNA complex was precipitated overnight with the antibodies of interest. Bound DNA was eluted by incubating the beads in elution buffer, purified, and amplified using primers flanking the Nrf2-binding site within the promoters of the genes encoding human GCLC and GSS. The oligonucleotide containing the transcription factorbinding site of the GCLC and GSS promoter was obtained from Bioneer (Seoul, Korea). The ChIP procedure was analyzed by PCR using human GCLC and GSS promoter-specific primers as follows: GCLC, forward (5<sup>ȝ</sup>-ATCTCCACGGTCCAGGTT-3ȝ) and reverse (5ȝ-CTCCCTCACCCTATCCATTT-3ȝ); GSS, forward (5ȝ-CTGGGAATAACCAGACACCTA-3ȝ) and reverse (5<sup>ȝ</sup>-CAGGTTCAAGCAATTCTCCTG-3ȝ). The cycle parameters were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min. The amplified products were resolved by electrophoresis on a 3% agarose gel, stained with RedSafe™ nucleic acid staining solution, and photographed under UV light using Image Quant™ TL analysis software. 

## *3.7. Luciferase Reporter Assay*

HaCaT cells were transiently transfected with 0.5 ΐg of the luciferase reporter and 0.2 ΐg of the ARE expression vector using Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, CA, USA). Co-transfection with 0.02 ΐg of pRL-TK Renilla reniformis luciferase served as a normalizing control. Luciferase assays were performed using the dual luciferase assay system (Promega, Madison, WI, USA). 

## *3.8. Detection of GSH*

For image analysis of GSH, cells were seeded in four-well chamber slides at a density of 

1 × 105 cells/mL. Sixteen hours after plating, cells were treated with 20 ΐM fucoxanthin and then irradiated with UVB 1 h later. After 12 h, 10 ΐM of CMAC was added to each well, and samples were incubated for an additional 30 min at 37 °C. After washing with PBS, the stained cells were mounted onto a chamber slide in  mounting medium. Images were collected on a confocal microscope using the LSM 5 PASCAL software (Carl Zeiss, Jena, Germany). In addition, the GSH concentration was measured using a BIOXYTECH GSH-400 assay kit (Foster City, CA, USA). 
