Figure 3: (a) Time courses of changes in the mRNA levels of heme oxygenase-1 (HO-1), ABCG2, and ABCB6 in HepG2 cells incubated with protoporphyrin IX (PpIX) and exposed to visible light. In dark conditions, HepG2 cells (1 106 cells) were first incubated with 10  M PpIX in Dulbecco’s modified Eagle’s medium at 3 C under 5% CO2 gas for 4 hours. Thereafter, the incubation medium was replaced with fresh incubation medium. Cells were then exposed to visible light for 90 min, where cell culture dishes containing HepG2 cells were placed on a light viewer (Hakuba Model 5700) in an incubation chamber (3 C, 5% CO2). Cells were subsequently incubated in the dark at 3 C under 5% CO2 gas for 22.5 hours. The whole incubation was performed for a total of 28 hours, as indicated in the figure. (b) As the control experiments without light exposure, HepG2 cells (1 106 cells) were incubated with 10  M PpIX in the same way as described above. PCR primers to quantitatively measure mRNA levels of HO-1, ABCG2, ABCB6, and GAPDH are described in [44]. (c) Schematic illustration for the activation of Nrf2 via three different pathways ((i), (ii), and (iii)). Pathway (i), under homeostatic conditions, Nrf2 is sequestered in the cytoplasm by the Keap1-Cul3 complex and rapidly degraded in a ubiquitin-proteasome-dependent manner. After an oxidative challenge (e.g., 1O2), oxidation of two reactive cysteine residues of Keap1 inhibits the ubiquitination reaction of Nrf2 mediated by the Keap1-Cul3 complex, which results in both cytoplasmic accumulation and nuclear translocation of Nrf2. Pathway (ii), activation of Nrf2 is mediated by protein kinases (PKs), such as p3 , PI3K, PERK, and PKC. Pathway (iii), under normal conditions, the chromatin structure of HO-1 is in a preactivation state, but transcription is repressed by Bach1. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. In the nuclei, the activated Nrf2 dimerizes with small Maf nuclear protein for effective binding to the ARE consensus sequence in the promoter region of the HO-1 gene.