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. Author manuscript; available in PMC: 2017 May 9.
Published in final edited form as: Cell Rep. 2017 Apr 11;19(2):255–266. doi: 10.1016/j.celrep.2017.03.041

Figure 5. Rfk knockdown results in altered expression of genes in metabolic pathways.

Figure 5

(A) mRNA levels of G6pc and Pck in mouse liver. mRNA levels were normalized to Tbp. Data are shown as means ± SEM (n=3, *: p<0.05 by two-way ANOVA). (B) Glucose levels in blood serum (left) and liver (right) at CT12. Data are shown as means ± SEM (n=3, *: p<0.05 by Student’s t-test) (C) Oxygen consumption rate assay in HepG2 cells. Cells were transfected with siRNA of Rfk or treated with 100 μM FAD for 24 hours. Data are shown as means ± SEM (n=6). Statistical significance was determined by two-way ANOVA. (D) Extracellular acidification rate assay in HepG2 cells. Cells were transfected with Rfk siRNA or treated with 100 μM FAD for 24 hours before recording fluorescence of a glycolysis probe. Data are shown as means ± SEM (n=5 for FAD treatment, n=9 for Rfk knockdown). Statistical significance was determined by two-way ANOVA. (E) Oxygen consumption rate assay in HepG2 cells transfected with siRNA of Rfk and/or CRY1/2. Data are shown as means ± SEM (n=9) (F) Extracellular acidification rate assay in HepG2 cells transfected with siRNA of Rfk and/or CRY1/2. Data are shown as means ± SEM (n=9) (G) mRNA levels of indicated metabolism-relevant genes in mouse liver. mRNA levels were normalized to Tbp. Data are shown as means ± SEM (n=3, *: p<0.05 by two-way ANOVA). (H) Proposed model of CRY regulation by FAD. The classical clock generates gene expression rhythms including Rfk and Nampt, which rhythmically synthesize FAD and NAD+. FAD binding to CRY prevents FBXL3-mediated CRY degradation, leading to stabilization. Time-dependent FAD synthesis modulates CRY expression rhythms and amplitudes, which regulate the gene expression rhythms of important genes for metabolism.