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. 2022 Feb 9;51:102261. doi: 10.1016/j.redox.2022.102261

Fig. 6.

Fig. 6

REV-ERBα regulates NRF2(Nfe2l2) transcription and the expression of its downstream target antioxidant genes in RPE cells. (A) mRNA expression of NRF2 (Nfe2l2) and its downstream target antioxidant genes (Sod1, Sod2, Cat) in the RPEs of 12-month-old Rev-erbα+/+ and Rev-erbα−/− mice. Real-time q-PCR results were normalized to 18S and then normalized again to the expression levels in Rev-erbα+/+ RPE cells. n = 6/group. (B) Western blot and quantification of NRF2, SOD1, SOD2, and catalase protein levels in 12-month-old Rev-erbα+/+ and Rev-erbα−/− RPEs, with a confirmation of REV-ERBα knockout. Protein sizes: nuclear NRF2 (110 kDa), SOD1 (20 kDa), SOD2 (20 kDa), catalase (60 kDa). n = 3 eyes/group. (C) Protein levels of NRF2, SOD1, SOD2 and catalase were assayed and quantified in the primary RPE cells from vehicle- and SR9009-treated mice after NaIO3 injection by Western blotting. n = 3 eyes/group. (D) A schematic diagram shows RORE/RevRE elements across the human NFE2L2 (NRF2) upstream regulatory region. The thick truncated lines mark the regions covered by 3 primer sets of interest (NFE2L2 #1 + 259∼+354, NFE2L2 #2–607∼-508, NFE2L2 #3–924∼-814). TSS: Transcription start site. (E) Chromatin-immunoprecipitation (ChIP) assays for binding of REV-ERBα with the regulatory region upstream of the NFE2L2 gene. The ChIP assay was conducted to analyze the local enrichment of REV-ERBα-associated ROREs across the upstream regulatory region and part of 5′UTR of the NFE2L2 gene in ARPE19 cells under four conditions: vehicle vs. SR9009 treatment and shControl vs. shREV-ERBα. In addition to probing for NFE2L2 #1, NFE2L2 #2, NFE2L2 #3, a pair of primers covering the RORE of the BMAL1 promoter was probed as a positive control. The relative fold enrichment was quantified by normalization to input, and then normalized against the levels in normal rabbit IgG pulled down groups. n = 6/group. (F) ChIP assay was conducted to analyze the local enrichment of DNA fragments containing the NFE2L2 #1 region in ARPE-19 cells with REV-ERBα, HDAC3 and NCOR1 antibodies pull down. The relative fold enrichment was quantified by normalization to input, and then against the levels in IgG group. n = 6/group. (G)Western Blot analysis of ARPE-19 cells transfected with either vehicle or p3Flag-REV-ERBα confirmed over-expression of flag-tagged REV-ERBα (Flag-REV-ERBα), with slightly higher MW than native REV-ERBα. (H) Luciferase assay was conducted in ARPE19 cells transfected with pGL3 (empty vector), pGL3-NFE2L2 #1 (+164∼+466), and pGL3-NFE2L2 #3 (−898∼-716) with vehicle vs. p3Flag-REV-ERBα, or vehicle VS. SR9009. Relative luminescence units (RLU) were quantified after normalizing to Renilla luciferase. n.s.: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001****: P < 0.0001. Error bars indicate SD.