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. 2016 Sep 13;24(3):462–473. doi: 10.1016/j.cmet.2016.07.024

Figure 5.

Figure 5

NRF2 Mediates the Effect of Redox Perturbation on Circadian Oscillations

(A) De novo HOMER motif analysis of the indicated sets of BMAL1/CLOCK peaks.

(B) Histogram of E-box and NRF2-like motif positions around BMAL1/CLOCK peaks bound only the 6AN condition.

(C) Immunoblot showing NRF2 nuclear accumulation in cells treated with 6AN or control (DMSO). U2AF65 is shown as loading control. Molecular weights (kDa) shown on right side of blots.

(D) Densitometric quantification of blots from (C) (mean ± SEM, n = 3; two-tailed Student’s t test, ∗∗p < 0.01).

(E) ChIP followed by quantitative real-time PCR of NRF2 following 6AN treatment or control (DMSO) (mean ± SEM, n = 3; one-tailed Student’s t test, ∗p < 0.05). HMOX1, heme oxygenase 1.

(F) mRNA accumulation of clock gene transcripts in Bmal1:luc U2OS cells following knockdown with 20 nM NRF2 siRNA or control (non-targeting siRNA #1). Bmal1:luc cells were synchronized 72 hr after transfection with a dexamethasone shock, and total RNA was collected after 24 hr incubation with 5 mM 6AN or control (DMSO) (mean ± SEM, n = 3; two-tailed Student’s t test; ∗∗∗p < 0.001, ∗∗p < 0.01). NS, not statistically significant by t test.

(G and H) Bioluminescence recordings of Bmal1:luc U2OS cells transfected with 20 nM NRF2 siRNA or control (non-targeting siRNA #1) combined with 6AN treatment at 1.25 mM (G) or control (DMSO) (H) (left; mean, n = 8). Quantifications of circadian period length of bioluminescence traces (right; mean ± SEM, n = 8; two-tailed Student’s t test; ∗∗p < 0.01).