Figure 2 : SREBP1c directly activates CRY1 gene expression.

From: SREBP1c-CRY1 signalling represses hepatic glucose production by promoting FOXO1 degradation during refeeding

Figure 2

(a,b) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-SREBP1c, as indicated. The levels of the CRY1 mRNA (a) and CRY1 protein (b) were determined using qRT-PCR with normalization to TBP mRNA levels and western blotting, respectively. Data are represented as mean ±s.d., N=3 for each group. *P<0.05, **P<0.01 (Student’s t-test). (c) Luciferase activity of the WT CRY1 promoter and 3XSRE mutant promoter were measured following co-transfection with expression plasmids encoding either SREBP1c or MOCK in HEK293T cells. Luciferase activity was normalized by β-gal activity. TSS, Transcription Start Site; SRE, Sterol Regulatory Elements. Data are represented as mean ±s.d., N=5 for each group. **P<0.01 (Student’s t-test). (d) ChIP assay, performed as described in Methods section, showing CRY1 promoter occupancy by SREBP1 in H4IIE cells. (e) Mouse primary hepatocytes were isolated from SREBP1c−/− and SREBP1c+/+ mice. With insulin (10 nM), the levels of SREBP1c and CRY1 protein were determined using western blotting. (f) SREBP1c−/− and SREBP1c+/+ mice were fasted for 24 h and then refed for 12 h. Both fasted and refed mice were sacrificed at ZT 3. The levels of SREBP1c and CRY1 mRNAs were determined by qRT-PCR and normalized to TBP mRNA levels. Data are represented as mean ±s.d., N=3–4 for each group. *P<0.05, **P<0.01 (Student’s t-test). See Supplementary Fig. 13 for original full immunoblot.