Figure 4: CRY1 regulates FOXO1 protein level.

(a,b) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-CRY1. The expression profiles of FOXO1 were analysed at the protein level (a) using western blotting and at the mRNA level (b) using qRT-PCR. Data are represented as mean ±s.d., N=4 for each group. *P<0.05 (Student’s t-test). (c,d) H4IIE cells were transfected with siCON or siCRY1. Immunocytochemical analysis (c) of endogenous FOXO1. DAPI, 4’, 6-diamidino-2-phenylindole. Scale bars, 10 μm. Endogenous FOXO1 and CRY1 protein levels were analysed using western blotting (d). (e,f) The expression patterns of FOXO1 protein in the liver of CRY1^+/+ and CRY1^−/− mice were analysed by western blotting (e) and qRT-PCR (f). Relative mRNA levels were determined using qRT-PCR and normalized to the levels of the TBP mRNA. Data are represented as mean ±s.d., N=3 for each group. *P<0.05, (Student’s t-test). (g) Expression of CRY1 and FOXO1 proteins in the liver of SREBP1c^+/+ and SREBP1c^−/− mice was analysed by western blotting. (h) H4IIE cells were co-transfected with siCRY1 and/or siFOXO1. Relative mRNA levels were determined using qRT-PCR and normalized to the cyclophilin mRNA level. Data are represented as mean ±s.d., N=3 for each group. ^#P<0.05, ^##P<0.01 versus siCRY1, *P<0.05, **P<0.01, ***P<0.001 versus siCON (Student’s t-test). See Supplementary Fig. 13 for original full immunoblot.