Fig. 7. Increased accumulation of Nrf2 in the nuclei of AREc32 cells exposed to the monofunctional inducer tBHQ. AREc32 cells were cultured in DMEM supplemented with antibiotics and exposed to DMSO or 10 mM tBHQ for 24 h. Nuclear extracts (20 mg) were subjected to 7% SDS/PAGE, and the level of Nrf2 protein was measured by Western immunoblotting. The standard (std) was 1 ng of recombinant his-mNrf2. The blots shown are representative of at least three separate experiments.

Fig. 8. Time course of ATRA inhibition of ARE activity. AREc32 cells were incubated with DMEM containing tBHQ (10 mM), ATRA (1 mM), or tBHQ (10 mM) plus ATRA (1 mM) for 3-24 h. Luciferase activity is expressed relative to DMSO (0.1% vol/vol) (control) activity. Values shown are mean ± SD. for significance determination.

Fig. 9. Effect of prior incubation with tBHQ on inhibition of ARE activity by ATRA. AREc32 cells were seeded in 96-well plates at 1.2 ´ 10⁴ cells per ell. After 24 h, the culture medium was replaced with fresh DMEM containing tBHQ (10 mM). After 2 h, ATRA (1 mM) was added into the DMEM containing tBHQ (10 mM). The cells were further incubated for 4 h before luciferase determination. Values are expressed as fold DMSO control and the data shown represent the results of triplicate determination from three separate experiments. **, P < 0.001

Fig. 10. Effect of 9-cis-RA, 13-cis-RA on ARE inhibition. AREc32 cells were seeded in 96-well plates at 1.2 ´ 10⁴ cells per well. After 24 h, the tBHQ (10 mM) and 9-cis-RA or 13-cis-RA were added concomitantly to the medium. After further 24 h, samples were assayed for luciferase activity. The value of luciferase activity of cells treated with 10 mM tBHQ alone (control) was taken as 100%. Values shown are mean ± SD.

Fig. 11. Reduction in RARa and RARg expression by using RNAi. AREc32 cells were seeded at 4 ´ 10⁵ cells per well in six-well plates, containing diluted RARaRNAi #1 or RARg RNAi (200 pmol per well) and Lipofectamine 2000 (10 ml per well) as described in Materials and Methods. Crude cell extracts were prepared from the cells transfected for 24 h and 48 h and separated by 10% SDS/PAGE. The depletion of RARa and RARg was confirmed by immunoblotting. (Lower) Actin was used as loading control.

Fig. 12. Reduction of Nrf2 binding to the ARE in the presence of ATRA. Nuclear extracts were prepared from AREc32 cells incubated with 10 mM tBHQ in the presence or absence of 1 mM ATRA for 24 h. Nuclear protein (100 m g) was incubated with biotinylated ARE oligonucleotide, and a pull-down assay performed. The pull-down beads were subjected to SDS/PAGE and immunoblotted with a specific Nrf2 antibody. The density of bands representing Nrf2 was quantified and the result shown is a representative of at least three independent experiments.