Fig 2: Core-clock manipulation affects EMT gene expression in SW480 and SW620 cells. Gene expression analysis of core-clock genes PER2, CRY1, NR1D1, CLOCK, and ARNTL as well as EMT-related genes in control and core-clock knockdown (shARNTL, shPER2 and shNR1D1, respectively) cell lines in SW480 (A) and SW620 (B) cells at 24 h after synchronization (n = 3, mean ± SEM). Heatmaps of Pearson correlations between each pair of core-clock and EMT-related genes for (C) SW480 and (D) SW620 shARNTL, shPER2 and shNR1D1 cells compared with the control cell line. * p < 0.05, ** p < 0.01, *** p < 0.001; two-tailed unpaired t-test.

Fig 3: Genetic activation of NRF2 perturbs circadian rhythmicity and regulates Nr1d1 gene expression.(A) Per2:Luc-driven bioluminescence from Wt and Keap1-/- MEFs. Average luminescence recordings are shown on the left. Amplitude and period length are expressed as an average ±standard deviation (n = 5). Results were analyzed using Student’s t-test; * indicates a p-value<0.05 relative to Wt. (B) Per2:Luc-driven bioluminescence from Wt MEFs stably expressing an empty vector (Wt) or a Nrf2 expression construct (CMV-Nrf2). Average luminescence recordings are shown on the left. Amplitude and period length are expressed as an average ±standard deviation (n = 3). Results were analyzed using Student’s t-test; * indicates a p-value<0.05 relative to Wt. (C) NRF2, NQO1, and NR1D1 protein in 50 µg of whole cell lysate from Wt and Wt +CMV-Nrf2 MEFs. ß-Actin, which was unchanged by exogenous Nrf2 expression, was used as a loading control. Values are expressed as the normalized average densitometry ±standard deviation (n = 3). Results were analyzed using Student’s t-test; * indicates a p-value<0.05 relative to the Wt. (D) Nqo1 and Nr1d1 gene expression, in Wt and Wt +CMV-Nrf2 MEFs. Expression values were determined by qPCR and normalized to Gapdh. Data is shown as the average fold-change ±standard deviation (n = 3) relative to the expression in Wt, which was set to 1. Results were analyzed using Student’s t-test; * indicates a p-value<0.05 relative to the Wt. (E) Nqo1 and Nr1d1 gene expression in Wt and Nrf2-/- MEFs treated with DMSO (Veh) or 100 µM D3T for 24 hr. Expression values were determined by qPCR and normalized to Gapdh. Data is shown as the average fold-change ±standard deviation (n = 3) relative to the expression in the Wt Veh, which was set to 1. Results were analyzed using a two-way ANOVA followed by Tukey’s multiple comparisons test; a indicates a p-value<0.05 relative to the Wt Veh control, b indicates a p-value<0.05 relative to treated Wt sample. (F) Chromatin immunoprecipitation-PCR using an NRF2 antibody to detect binding of NRF2 to the indicated gene promoters. Wt MEFs were treated with DMSO (Veh) or 100 µM D3T for 24 hr. Putative ARE sequences can be found in Supplementary file 2 and their location in the respective promoter regions can be found in Figure 3—figure supplement 1.

Fig 4: Jet lag disrupted the rhythmicity of circadian clock genes. The total mRNA was isolated from the colon tissue and qPCR was carried out using different primers. Gapdh was used as a normalized control. (A) The qPCR assays of the core clock genes in mice in the jet-lag and control groups at different time points (six mice per group). (B) Western blot analysis of the core clock genes at zeitgeber time (ZT)4 and ZT16 (six mice per group). (C) The relative expression of Bmal1, Clock, Per1, Per2, Cry1, Cry2, and Nr1d1 normalized with Gapdh (six mice per group). Presented data are expressed as mean ± SD. *P < 0.05 vs control mice at individual time points (t-test).

Fig 5: REV-ERB deletion leads to exaggerated activation of CFs. (A) Representative phase-contrast and GFP fluorescence images of CFs from Nr1d1/2 fl/fl mouse infected with Ad-Cre-GFP or Ad-GFP virus. (B) Relative mRNA expression of Nr1d1 and Nr1d2 with qRT-PCR primers spanning loxP sites n=3. (C) Representative images of immunostaining of a-SMA (Red) and DAPI (Blue) for Nr1d1/2 fl/fl (control) and DKO CFs with or without TGFß-1 treatment. (D) Quantification of stress fiber positive cells in Nr1d1/2 fl/fl (control) and DKO CFs with or without TGFß-1 treatment n=6. (E) Relative mRNA expression of Acta2, Col1a1, and Fn1 in Nr1d1/2 fl/fl and DKO CFs with or without TGFß-1 treatment n=6 to 18. (F) Representative images and quantification of the wound healing assay at different time points of the Nr1d1/2 fl/fl and DKO CFs, n = 3, data are presented as mean ± SEM. (G) BrdU assay. The absorbance of BrdU incorporation was measured at 450 nm after 6 h incubation for Nr1d1/2 fl/fl and DKO CFs, n = 8. (H) CCK-8 assay. The absorbance was measured at 450 nm for Nr1d1/2 fl/fl and DKO CFs after 5h incubation with the WST-8 solution, n = 5. *p = 0.05, ***p < 0.001, and ****p < 0.0001 by one-way ANOVA (H), two-way ANOVA (D–F), and 2-sided Student’s t-test (B and G). Data are presented as mean ± SEM.