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[Effect of DRB/alpha-Amanitin on localization of Nrf2 in A549 cells]
Li-yan Qu1, Yan-ling Jiang, Xiu-wen Tang
1Department of Biochemistry and Genetics, College of Medicine, Zhejiang University, Hangzhou, China.
Objective:
To investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.
Methods:
A549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.
Results:
The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.
Conclusion:
DRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.
Insights
Transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin reduce Nrf2 and its target gene expression. These inhibitors do not significantly alter the nuclear localization of Nrf2.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the antioxidant response.
- Transcriptional inhibitors like DRB and alpha-Amanitin are used to study gene expression regulation.
- Understanding Nrf2's nuclear localization is crucial for comprehending its role in cellular defense.
Purpose of the Study:
- To investigate the impact of DRB and alpha-Amanitin on Nrf2 nuclear localization.
- To analyze the effects of these inhibitors on the expression of Nrf2 and its downstream targets.
Main Methods:
- A549 cells were treated with DRB or alpha-Amanitin.
- Western blotting was employed to detect protein expression levels.
- Laser scanning confocal microscopy was used to determine Nrf2 localization.
Main Results:
- DRB and alpha-Amanitin treatment led to decreased expression of Nrf2 and its target genes (HO-1, NQO1, AKR1C).
- SC35 expression was upregulated, while RNA Pol II expression was downregulated.
- No significant changes were observed in the nuclear localization of Nrf2.
Conclusions:
- DRB and alpha-Amanitin effectively downregulate Nrf2-mediated gene expression.
- These transcriptional inhibitors do not appear to affect the nuclear localization of Nrf2.